LC Technical NoteLT156

GL Sciences Inc.

Sample Preparation of Peptides and Proteins Using MonoSpin C18- Obtain High Recovery with Ethanol Elution -Reversed-phase HPLC is used for the quantitative analysis of proteins. Proteins are separated based on thedifference of hydrophobicity. Sample preparation is necessary when analyzing biological samples to reduce theaffect from matrix.
Purification of biological samples using revers-phase solid phase extraction requires acetonitrile or methanol
contained solvent for analyte elution. However, there is a case high recovery can’t be obtained due to a few
factors: slow partition, precipitation by high concentration of organic solvent and strong adsorption to packing
material.
In this note, MonoSpin, uniquely suited for handling small amount samples, is used to purify the proteins.
During the process, changes in selectivity and recovery are seen due to the usage of different kind of organicsolvent.
(Y. Yui, S.Ota)

What is MonoSpin?
MonoSpin SPE centrifugal spin columns
were developed to improve concentrationof small-volume sample preparation. The
low-pressure, high-flow, and low-liquidretention properties of GL Sciences’monolith silica technology makes it
uniquely suited for handling small amountsamples.

Monolith
structure

Centrifuge Operation MonoSpin

Sillica Monolith
DiskOperation of MonoSpin C18
Ethanol is used for elution

Spin

Spin

Spin

Spin

Spin

2,000×g
30 sec.

2,000×g
30 sec.

2,000×g
30 sec.

2,000×g
1 min.

2,000×g
1 min.

Conditioning
CH3CN 300 μL→50 % CH3CN in H2O
300 μL→0.2 % TFA in H2O
300 μL

Adsorption

Rinsing

1st Elution

2nd Elution

Sample300 μL

0.2% TFA in H2O
300 μLC2H5OH /H2O/TFA=60/40/0.1
300 μLC2H5OH /H2O/TFA=60/40/0.1
300 μL

Adsorption

Rinsing

1st Elution

2nd Elution

Samples
1.RibonucleaseA (Mw 13,700)
2.Insulin (Mw 5,800)
3.Lysozyme (Mw 14,307)
4.BSA (Mw 66,000)
5.Myoglobin (Mw 17,500)
0.05 mg/mL eachin （0.1%TFA in H2O）GL Sciences LC Technical Note
Conditions
Column：Eluent：

Chromatogram
1.RibonucleaseA (Mw 13,700)
2.Insulin (MW 5,800)
3.Lysozyme (MW 14,307)
4.BSA (MW 66,000)
5.Myoglobin (MW 17,500)

Flow rate：
Col.Temp：
Detection：Injection Vol：

2STD

InertSustainBio C18 （1.9 μm, 100× 2.1 mm I.D.）
A) 0.1 % TFA in H2OB) 0.1 % TFA in CH3CN
A/B=72/28 - 20min - 45/55 - 0.1 min - 72/28 – 5 min
0.2 mL/min
40 ºC
UV 220 nm
2 μL

35Volts
0.02

10.00

4010Time (min)1st Elution

220

35Volts
0.02

10.00

4010Time (min)

2nd Elution

20

Volts
0.02

4Enlarged view

0.00

4010
Time (min)20

GL Sciences LC Technical Note
Recovery (%)
Recovery (n=3) is calculated by the peak area. Only BSA isn’t recovered enough compared toother proteins. However, it makes possible to increase recovery by eluting twice.

Purification
Process
Compounds

Recovery (%)

Adsorption

Rinsing

1st Elution

2nd Elution

RibonucleaseA

00101.00

Insulin00

104.90Lysozyme

3.60

91.80BSA

11.5

0

75.7

7.8

Myoglobin

00

99.90

Also, poor recovery is seen when eluting with acetonitrile or methanol.
Recovery rate changes depending on elution solvent.

Acetonitrile elution
Acetonitrile is used at 1st and 2nd elution instead of ethanol.
Purification
ProcessAdsorption
Compounds

Recovery (%)

Rinsing

1st Elution

2nd Elution

RibonucleaseA00

12.8

8.4

Insulin

00

107.7

2.0

Lysozyme

2.5093.8

8.5

BSA

9.7060.9

6.3

Myoglobin

0097.3

7.8

Methanol elution
Methanol is used at 1st and 2nd elution instead of ethanol
Recovery (%)

Purification
Process
Compounds

Adsorption

Rincing

1st Elution

2nd Elution

RibonucleaseA

00

83.2

8.7

Insulin

00

94.8

0.9

Lysozyme

4.6000BSA

12.0000

Myoglobin

0001.3

GL Sciences LC Technical Note
Products

MonoSpin C18Cat. No. 5010-21700 (50 pcs)
Cat. No. 5010-21701 (100 pcs)
*We have trial kit for initiating your analysis.

MonoSpin Customize 20：

You may choose 2 kinds of MonoSpin (10 pcs each)
Trypsin is an exception
Cat. No. 5010-01001

MonoSpin Trial 1 :Appropriate for pestiside (C18, SCX, SAX, TiO /10 pcs)
Cat. No. 5010-21740

MonoSpin Trial 2 :Appropriate for sugars and hydrophilic compounds. (C18, Amide, CBA, NH2 /10 pcs）
Cat. No. 5010-21741

MonoSpin Trial 3 :Appropriate for ionic compounds (SCX, SAX, CBA, NH2 /10pcs）
Cat. No. 5010-21742

HPLC・LC/MS Column

InertSustainBio C18
Rapid Separations of Proteins and Peptides

Column sectional view
StainlessFeatures
・Separation of compounds from small to large molecules
・A radically new Steel-Coated-PEEK hardware preventing adsorption of peaks
・Suitable for analyzing Phosphate compounds

PEEK

Packing material

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly. We reserve theright to amend this information or data at any time and without any prior announcement.

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