Chromatography
Analysis
Application Note: CHROM307
Separation and Purification of α Chymotrypsinogen and
Cytochrome C to Compare OmniSep™ A-CM Dash and
CM Sepharose® FF Columns
Introduction
For the comparison of OmniSep A-CM Dash and CM Sepharose FF the sample purified was a mixture of α
Chymotrypsinogen and Cytochrome C.
Chymotrypsinogen is a proteolytic enzyme and is a precursor of chymotrypsin a digestive enzyme. It is
constructed of a single polypeptide chain consisting of 245 amino acid residues and is synthesized in the
cells of the pancreas and stored. The cell when stimulated by either a hormonal signal or a nerve impulse
empties the stored contents into a duct leading into the duodenum.
Chymotrypsinogen must be inactive until it gets to the digestive tract. This prevents damage to the pancreas
and other organs.
Cytochrome C proteins are found loosely associated with the inner membrane of the mitochondrion.
Cytochrome C is a small (molecular weight about 12,000 Dalton’s), highly soluble protein, unlike other
cytochromes, with a solubility of about 100 g/L and is an essential component of the electron transport
chain, where it carries one electron. It is capable of undergoing oxidation and reduction, but does not bind
oxygen.
Cytochrome C is a highly conserved protein across many species, found in animals, plants, and many
unicellular organisms. Its structure consists of a chain of 100 amino acids. The cytochrome c molecule has
been studied in evolutionary biology; its amino acid sequence is conserved in mammals differing by only a
few residues. For example, the sequences of cytochrome c in humans are identical to that of chimpanzees
(our closest relatives).
Two identical columns were used for the evaluation, separation and purification. They were packed in the
OmniSep manufacturing suite utilising OmniFit Benchmark Columns (Part Number – 006BCC-15-15-AF) - 15
x 150mm with one adjustable and one fixed end fitting. These columns have a column volume of ~18mL
when packed to a bed height of 10cm.
One column was packed with OmniSep A-CM Dash and the other with CM Sepharose FF.
When the packing process was completed the columns were run sequentially on an ÄKTA® using identical
conditions:
Separation Conditions
Eluent A:
20mM Tris Buffer pH 8.2
Eluent B:
20mM Tris Buffer + 0.5M NaCl pH 8.2
Linear Gradient to 50%B
Flow rate:
5ml/min
Columns were equilibrated for 3 column volumes (CV), 12 minutes
Gradient:
Time
0
04
24
%B
0
0
50
0.2mL sample injected
Results
Data was collected on the ÄKTA using the ÄKTA Unicorn software version 5.0 and the chromatograms
integrated to provide values for N – Column Efficiency in plates per metre, As – Column Asymmetry and
Plate Height.
Chromatograms and chromatographic reports from the ÄKTA are included on the right (Fig. 1 and Fig.2)
and show no significant difference in the separation profiles between the OmniSep A-CM Dash and CM
Sepharose FF columns, with similar retention times, efficiencies and asymmetries.
The OmniSep A-CM Dash column is a “plug and play” alternative for the CM Sepharose FF column.
1. Sample Volume and Type:
Alpha Cymotrypsinogen and Cytochrome C 10m g/ml
2. Column:
OmniSep CM
3. Eluent A:
TRIS 20mM pH 8.2
4. Eluent B:
TRIS 20mM + 0.5M NaCl pH 8.2
5. Remarks:
-
No.
Peak Name
Ret Width at Half
Height
Plate Height
Plates/Meter
10% Left
10% Right
Asymmetry
MIN
MIN
CM
N/m
MIN
MIIN
1.
-8.01
067
0.01258
7951
7.55
8.66
1.40
2.
-12.46
1.47
0.02508
3987
11.24
13.86
1.15
Diba QC OmniSep A Dash Application 15mm011:10_UV1_280nm@01,BASEM
Diba QC OmniSep A Dash Application 15mm011:10_UV1_280nm
12.46
mAU
OmniSep
A-CM
Dash
Column
40.0
8.01
30.0
20.0
10.0
0.0
0.0
5.0
10.0
15.0
min
Figure 1: UV chromatogram of α Chymotrypsinogen and Cytochrome C samples on OmniSep A-CM Dash
1. Sample Volume and Type:
Alpha Chymotrypsinogen and Cytochrome C 10m g/ml
2. Column:
Sepharose CM 15 x 100mm
3. Eluent A:
TRIS 20mM pH8.2
4. Eluent B:
TRIS 20mM + 0.5M NaCl pH8.2
5. Remarks:
-
No.
Peak Name
Ret Width at Half
Height
Plate Height
Plates/Meter
10% Left
10% Right
MIN
MIN
CM
N/m
MIN
MIIN
1.
-7.40
0.55
0.00989
10108
6.96
7.98
1.31
2.
-11.36
1.22
0.02083
4800
10.17
12.53
0.99
Diba QC OmniSep A Dash Application 15mm009:10_UV1_280nm@01,BASEM
Diba QC OmniSep A Dash Application 15mm009:10_UV1_280nm
mAU
40.0
30.0
GE CM Sepharose Column
Asymmetry
11.36
7.40
20.0
10.0
0.0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Figure 2: UV chromatogram of α Chymotrypsinogen and Cytochrome C samples on CM Sepharose columns
14.0 min
Trademarks
OmniSep™ is a trademark of Diba Industries Incorporated.
Sepharose® is a registered trademark of GE Healthcare Life Sciences.
ÄKTA™ is a trademark of GE Healthcare Life Sciences.
Available worldwide exclusively through
UNITED KINGDOM
Kinesis Ltd
Tel: +44 (0)1480 212122
Fax: +44 (0)1480 212111
E-mail: [email protected]
Web: kinesis.co.uk
GERMANY, SWITZERLAND
& AUSTRIA
Kinesis GmbH (formerly Abimed)
Tel: +49 (0)2173 89 05-0
Fax: +49 (0)2173 89 05-77
Email: [email protected]
Web: kinesisgmbh.de
Manufactured by
USA & CANADA
Kinesis Inc
Tel: (518) 289-5817
Toll free in USA: (866) 934-6353
Fax: (518) 289-5818
Email: [email protected]
Web: kinesis-usa.com
AUSTRALIA & NEW ZEALAND
Kinesis Australia Pty Ltd
Tel: +61 (0)7 3829 3996
Fax: +61 (0)7 3829 3997
Email: [email protected]
Web: kinesis-australia.com.au
EUROPE
Diba Industries Ltd.
2 College Park
Coldhams Lane
Cambridge, CB1 3HD, UK
Tel: +44 (0) 1223 472801
Fax: +44 (0) 1223 416787
[email protected]
www.omnisep.com
www.dibaind.com