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MICROCON®

Centrifugal Filter Devices

User Guide

For research use only,not for use in diagnostic procedures

Notice

The information in this document is subjectto change without notice and should not beconstrued as a commitment by MilliporeCorporation. Millipore Corporation assumesno responsibility for any errors that mayappear in this document. This manual isbelieved to be complete and accurate at thetime of publication. In no event shallMillipore Corporation be liable for inciden-tal or consequential damages in connectionwith or arising from the use of this manual.

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

®

99394, Rev. M, 06/05

© 2005. Millipore Corporation. All rights reserved.Millipore, Amicon, Centricon, Centriprep, Centriplus,Microcon, and Ultracel are registered trademarks ofMillipore Corporation.Alconox and Terg-A-Zyme are registered trademarks ofAlconox, Inc.Lubrol is a registered trademark of Imperial ChemicalIndustries PLC.Nonidet is a trademark of Royal Dutch/Shell Group.Triton is a registered trademark of Union CarbideCorporation.Tween is a registered trademark of ICI Americas Inc.

Contents

Introduction .......................1Diagram of MicroconCentrifugal Filter Device .................... 4

Intended Use ......................5Applications ..................................... 5

Equipment Required ...........6

Materials Supplied .............6

Limitations .........................6Maximum Centrifugal Force .............. 6Centrifugation Guidelines ................. 7Prerinsing Devices ............................ 8Spinning to Dryness .......................... 8Storage ............................................ 8Chemical Compatibility ..................... 9

ii www.millipore.com

How to Use the MicroconCentrifugal Filter Device .....11

Performance ....................15Flow Rate ....................................... 15Typical Protein Recovery ................. 16Retention and Recovery .................. 17Desalting/Diafiltration .................... 18

How to QuantitateRecoveries .......................18

Specifications ...................19Materials of Construction ................ 19

Ordering Information .......20

Technical Assistance .........21

Support Documents on theInternet ............................21

Standard Warranty ..........22

IntroductionMicrocon centrifugal filter devices simplyand efficiently concentrate and desalt mac-romolecular solutions of 10–500 µL usingany centrifuge that can accept 1.5 mLtubes. For best performance, use a centri-fuge with a fixed angle rotor. If using Mi-crocon devices with Ultracel® YM-100membrane to process DNA or RNA, a vari-able-speed centrifuge is required.

With a medical-grade silicone rubberO-ring sealing the membrane, Microconcentrifugal filter devices can be spun asmany as three times. See the “ChemicalCompatibility” section for solvent resis-tance.

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Introduction, continuedTo prevent mix-ups in the lab, eachMicrocon centrifugal filter device’ssample reservoir is color coded. TheMicrocon product line currently includesdevices with the following Ultracel YMmembranes:■ YM-3 (yellow top) — 3,000 Nominal

Molecular Weight Limit (NMWL)■ YM-10 (green top) — 10,000 NMWL■ YM-30 (clear top) — 30,000 NMWL■ YM-50 (rose top) — 50,000 NMWL■ YM-100 (blue top) — 100,000 NMWL

Ultracel low-binding, anisotropic, hydro-philic regenerated cellulose membrane isused in other high recovery centrifugalfilter devices including Centricon®, Cen-tricon Plus-20, Centricon Plus-70,Centriprep®, and Centriplus® devices.Because the same membrane is used inmany of Millipore’s centrifugal filter de-vices, scale up for higher volumes sim-ply by switching to a larger device.

Microcon Centrifugal Filter Devices 3

Introduction, continuedThe low-adsorption characteristics of theUltracel YM membrane and the device’scomponent parts, together with an in-verted recovery spin, combine to yieldunusually high recovery rates—typically> 95% of the sample, with concentrationfactors as high as 100×. The results ob-tained are consistent, assuring easy repro-ducibility from experiment to experiment.

Typical processing time is just 12–70 min-utes. Deadstop prevents spinning to dry-ness and potential sample loss.

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Diagram of Microcon CentrifugalFilter Device

Color codes

Yellow — Ultracel YM-3 membraneGreen — Ultracel YM-10 membraneClear — Ultracel YM-30 membraneRose — Ultracel YM-50 membraneBlue — Ultracel YM-100 membrane


Intended UseMicrocon centrifugal filter devices arefor research use only, not for use in di-agnostic procedures.

ApplicationsA full range of membrane cut-offs giveMicrocon centrifugal filter devices greatflexibility in performing a variety offunctions in addition to standardconcentration/desalting operations.Other uses include: buffer exchange; re-moval of primers, linkers, or molecularlabels from a reaction mix; and proteinremoval prior to HPLC. Microcon cen-trifugal filter devices provide a fast, effi-cient means for concentrating RNA/DNAsamples, either single- or double-stranded. The nucleotide cut-offs (NCO)listed in the “Centrifugation Guidelines”section can be used as a guide in select-ing the appropriate Microcon model foreach application.

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Equipment RequiredAny centrifuge that can properly accom-modate 1.5 mL microcentrifuge tubes isacceptable, although fixed-angle rotorsare preferred. If using a Microcon de-vice with Ultracel YM-100 membrane toprocess DNA or RNA, a variable-speedcentrifuge is required.

Materials SuppliedTwo vials with attached sealing caps areincluded with each centrifugal filter de-vice. During operation, one vial is usedto collect filtrate, the other to recoverretentate.

LimitationsMicrocon components are not autoclav-able. Do not operate above the follow-ing limits. Excessive g-force may resultin leakage or damage to the device.

Maximum Centrifugal Force■ Ultracel YM-3, YM-10, YM-30, and

YM-50 membrane: 14,000 × g

■ Ultracel YM-100 membrane for pro-teins: 14,000 × g

■ Ultracel YM-100 membrane for RNA/DNA samples: 500 × g

Microcon Centrifugal Filter Devices 7Ce

ntrif

ugat

ion

Gui

delin

es fo

r Mic

roco

n D

evic

esU

ltrac

el Y

MCo

lor

Mem

bran

eN

COM

axim

umSp

in T

imes

*M

embr

ane

Code

NM

WL

SSD

Sg-

Forc

e25

°C*

*Y

M-3

Yel

low

3,00

010

1014

,000

100

YM

-10

Gre

en10

,000

3020

14,0

0030

YM

-30

Cle

ar30

,000

6050

14,0

0012

YM

-50

Rose

50,0

0012

510

014

,000

12Y

M-1

00B

lue

100,

000

300

125

14,0

0012

500¹

12N

MW

L: N

omin

al m

olec

ular

wei

ght l

imit

in D

alto

ns (pro

tein

s)N

CO

: Nuc

leot

ide

cut-of

f for

RN

A/D

NA

(SS

=si

ngle

stran

ded,

DS

=do

uble

stran

ded)

*Tim

e in

min

utes

; 500

µL

sam

ple

s re

duce

d to

10

µL.

**Sa

mple

con

cent

ratio

n us

ing

Mic

roco

n de

vice

s at

4 °

C ty

pic

ally

take

s tw

ice

as lo

ng a

s th

at a

t 25

°C.

¹For

RN

A/D

NA

sam

ple

s us

ing

Mic

roco

n de

vice

s w

ith U

ltrac

el Y

M-1

00 m

embr

ane,

the

reco

mm

ende

d g-

forc

eis

500

×g.

NO

TE:

DN

A s

ample

con

cent

ratio

n us

ing

Mic

roco

n de

vice

s ge

nera

lly req

uire

s sh

orte

r sp

in ti

mes

. For

exam

ple

, at 2

5 °C

, a 1

0 µL

fina

l vol

ume

of a

n RN

A/D

NA

sam

ple

can

be

achi

eved

in a

Mic

roco

n de

vice

with

Ultr

acel

YM

-30

mem

bran

e in

just

8 m

inut

es.

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Prerinsing DevicesMicrocon ultrafiltration membranes con-tain trace amounts of glycerin. If glyc-erin interferes with analysis, spin-rinsedevices with deionized water or 0.1 NNaOH until no more interference is de-tected. If using 0.1 N NaOH, spin-rinsedevices with deionized water until pH offiltrate is neutral.

NOTE: Do not allow membranes to dryout before use.

Spinning to DrynessExtended centrifugation (2–3 times longerthan guidelines) can lead to dryness. Ifthis should occur, add at least 10 µL ofbuffer to the sample reservoir, agitategently for 30 seconds, then proceed withrecovery.

StorageFor best results, store all unused Microconcentrifugal filter devices at room tempera-ture.


Chemical CompatibilityThe solutions listed in this section havebeen evaluated for chemical compatibilityin Microcon devices containing Ultracel YMmembranes. Contact with some organicchemicals may cause leaching from compo-nent parts. If leaching is suspected, run sol-vent blanks before performing assays.

Test conditions: 6-hour exposure at 25 °C.

Common Acids/AlkalisAcetic acid (50%) ✓Ammonium hydroxide (10%) ✓Formic acid (50%) ✓Formic acid (70%) ✗Hydrochloric acid (1.0 N) ✓Hydrochloric acid (0.01 N) ✓Lactic acid (50%) ✓Sodium hydroxide (0.1 N) ✓Sodium hydroxide (2.5 N) ✗Sulfuric acid (3%) ✓Sulfuric acid (30%) ✗Trichloroacetic acid (10%) ✓Trifluoroacetic acid (30%) ✓

Organic Solvents/MiscellaneousAcetone ✗Acetonitrile (20%) ✓Alconox® detergent (1%) ✓Ammonium sulfate (saturated) ✓Benzene ✗Carbon tetrachloride ✗

✓ = Acceptable ✗ = Not Recommended ? = Questionable

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Chemical Compatibility, continued

Organic Solvents/MiscellaneousCHAPS (100 mM) ✓Chloroform ✗Diethyl pyrocarbonate (0.2%) ✓Dimethyl formamide ✗Dimethyl sulfoxide (5%) ✓Dioxane ✗Ethyl acetate ✗Formaldehyde (5%) ✓Formamide ✗Glycerin ✓Guanidine HCL (6 M) ✓Guanidine thiocyanate (0.5 M) ✓Guanidine thiocyanate (5 M) ✗Lubrol® PX emulsifier (0.1%) ✓Mercaptoethanol (0.1 M) ✓Nonidet™ P-40 surfactant (2%) ✓Phenol (1%) ✓Phosphate buffer (1 M, pH 8.2) ✓Polyethylene glycol ✓Pyridine ✗Sodium carbonate (20%) ✓Sodium chloride (2M) ✓Sodium deoxycholate (5%) ✓Terg-A-Zyme® detergent (1%) ✓Tetrahydrofuran ✗Toluene ✗Tris buffer (1 M, pH 8.2) ✓Triton® Z-100 surfactant (5 mM) ✓Tween®-20 surfactant (0.1%) ✓Urea (8 M) ✓

✓ = Acceptable ✗ = Not Recommended ? = Questionable


How to Use the MicroconCentrifugal Filter Device1. Insert Microcon sample reservoir into

vial.

2. Pipette solution into sample reservoir(0.5 mL maximum volume), withouttouching the membrane with the pi-pette tip. Seal with attached cap.

3. Place assembly in a compatible cen-trifuge (described in the “EquipmentRequired” section) and counterbal-ance with a similar device.

NOTE: When placing the assembleddevice into the centrifuge ro-tor, align the cap strap to-ward the center of the rotor.

Assembled device during concentration spin


How to Use the MicroconCentrifugal Filter Device,

continued4. Spin using the “Centrifugation Guide-

lines” for correct spin times andspeeds.

5. Remove assembly from centrifuge.Separate vial from sample reservoir.

Individual components separated after spinning



continued6. Place sample reservoir upside down

in a new vial, then spin 3 minutes at1000 × g (or pulse briefly) to transferconcentrate to vial.

Retentate transfer during recovery spin



continued7. Remove from centrifuge. Separate

sample reservoir. Snap sealing capshut to store product for later use.

Filtrate and retentate in sealable storage vials


PerformanceTests using Microcon centrifugal filter de-vices have characterized flow rate, reten-tion and recovery for several well-knownmaterials. These results, summarized inthe “Typical Protein Recovery” section,may help when estimating concentratorperformance with other solutes. Actualperformance, however, depends upon thenature of the specific solute under study.

The centrifugal forces presented hereare measured at the base of the filtratecup. Calculate centrifugal force usingthis formula:

RCF = 1.118 × 10-6 × radius × (RPM)2

radius = distance in millimeters fromcenter of rotation to base offiltrate vial

Flow RateFactors affecting flow rate include solutetype and concentration, the starting vol-ume, relative centrifugal force, angle ofcentrifuge rotor, membrane type (nominalmolecular weight limit) and temperature.

16 www.millipore.comTy

pica

l Pro

tein

Rec

over

y%

Ret

enta

te R

ecov

ery

by M

odel

Ultr

acel

YM

mem

bran

e

Solu

te/C

once

ntra

tion

Nom

inal

MW

310

3050

100

Bov

ine

IgG

Fra

ctio

n II

(1

mg/

mL)

156,

000

9595

9595

95

Bov

ine

seru

m a

lbum

in (

1 m

g/m

L)67

,000

9595

9590

50

Ova

lbum

in (

1mg/

mL)

45,0

0095

9595

9010

α-C

hym

otry

psin

ogen

(1

mg/

mL)

25,0

0095

9595

802

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c (0

.25

mg/

mL)

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9590

101

Prot

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(1

mg/

mL)

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103

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65

21

41


Retention and RecoveryThe anisotropic, hydrophilic Ultracel YMmembranes in Microcon centrifugal filterdevices are characterized by a nominalmolecular weight limit (NMWL), i.e., theability to retain molecules above a speci-fied molecular weight. While membraneretention is most closely related to mo-lecular size and shape, molecular weighthas proven to be a more convenient pa-rameter to use for most applications.The membrane’s NMWL is based on re-sults achieved with well-known solutesbut may not hold true for all solutes. Inparticular, solutes with molecularweights close to the membrane cut-offmay be partially retained.

Low solute recovery in the retentate mayindicate possible adsorptive losses and/or solute passage through the mem-brane. Adsorptive losses in Microcon de-vices depend upon several factors in-cluding: solute concentration, the hydro-phobic nature of the solute, operatingtemperature, length of time the solute isin contact with component surfaces,sample composition, and pH.


Desalting/DiafiltrationMicrocon centrifugal filter devices areuseful in a variety of applications, includ-ing desalting and buffer or solvent ex-change. To remove low molecular weightcontaminants using Microcon centrifugalfilter devices, concentrate repeatedly,then reconstitute with the appropriatesolvent. Typically two spins, each con-centrating the sample 20-fold, will pro-vide 95% exchange of buffers or removalof low-molecular-weight contaminants.

How to QuantitateRecoveriesTo calculate percent filtrate and reten-tate recovery, use the formulas below.

% retentate recovery = 100 ×

% filtrate recovery = 100 ×

Wr = total weight of retentate before assay

Wo = weight of starting material

Wf = weight of filtrate

Cr = retentate concentration

Co = starting material concentration

Cf = filtrate concentration

Wf × Cf

Wo × Co

Wr × Cr

Wo × Co


SpecificationsMaximum initial sample volume: 500 µL

(0.5 mL)

Typical final concentrate volume: 5–15 µL

Maximum relative centrifugal force:

Ultracel YM-3, YM-10, YM-30 & YM-50membranes: 14,000 × gUltracel YM-100 membrane: 14,000 × g for proteins 500 × g for RNA/DNA

Active membrane surface area: 0.32 cm2

Hold-up volume: 10 µL (unrecoverablevolume trapped beneath membrane)

Diameter: 12.3 mm

Length, concentration mode: 45.0 mm(includes concentrator in capped filtratevial)

Length, recovery mode: 48.2 mm(includes concentrator and retentate vial)

Materials of ConstructionMembrane: Ultracel YM regeneratedcellulose membraneSample reservoir: polycarbonateMembrane support base: acetalFiltrate/retentate vial: polypropyleneO-ring: medical-grade silicone rubber


Ordering InformationThis section lists the catalogue numbersfor Microcon centrifugal filter devices. Seethe Technical Assistance section for infor-mation about contacting Millipore. Youcan also buy Millipore products on-line atwww.millipore.com/purecommerce.

NMWL Units ProductMembrane & Color per Pack Number

Ultracel YM-3 3,000 8 42420Yellow Top 24 42403

100 42404

Ultracel YM-10 10,000 8 42421Green Top 24 42406

100 42407

Ultracel YM-30 30,000 8 42422Clear Top 24 42409

100 42410

Ultracel YM-50 50,000 8 42423Rose Top 24 42415

100 42416

Ultracel YM-100 100,000 8 42424Blue Top 24 42412

100 42413


Technical AssistanceFor more information, contact the Milli-pore office nearest you. In the U.S., call1-800-MILLIPORE (1-800-645-5476).Outside the U.S., see your Milliporecatalogue for the phone number of theoffice nearest you or go to our web siteat www.millipore.com/offices for up-to-date worldwide contact information.You can also visit the tech service pageon our web site at www.millipore.com/techservice.

Support Documents onthe InternetMillipore maintains a library of currentprotocol notes and other product informa-tion for Microcon centrifugal filter deviceson our web site: www.millipore.com/publications.


Standard WarrantyMillipore Corporation (“Millipore”) warrantsits products will meet their applicable pub-lished specifications when used in accordancewith their applicable instructions for a periodof one year from shipment of the products.MILLIPORE MAKES NO OTHER WARRANTY,EXPRESSED OR IMPLIED. THERE IS NOWARRANTY OF MERCHANTABILITY ORFITNESS FOR A PARTICULAR PURPOSE. Thewarranty provided herein and the data, specifi-cations and descriptions of Millipore productsappearing in Millipore’s published cataloguesand product literature may not be altered ex-cept by express written agreement signed byan officer of Millipore. Representations, oral orwritten, which are inconsistent with this war-ranty or such publications are not authorizedand if given, should not be relied upon.

In the event of a breach of the foregoing war-ranty, Millipore’s sole obligation shall be to re-pair or replace, at its option, the applicableproduct or part thereof, provided the customernotifies Millipore promptly of any such breach.If after exercising reasonable efforts, Milliporeis unable to repair or replace the product orpart, then Millipore shall refund to the cus-tomer all monies paid for such applicableproduct or part. MILLIPORE SHALL NOT BELIABLE FOR CONSEQUENTIAL, INCIDEN-TAL, SPECIAL OR ANY OTHER DAMAGESRESULTING FROM ECONOMIC LOSS ORPROPERTY DAMAGE SUSTAINED BY ANYCUSTOMER FROM THE USE OF ITS PROD-UCTS.