Reversed Phase Analysis

The relation between sample load and theoretical plate number for RSpak RP18-415 (a column for reversed phase chromatography) is shown. The theoretical plate number does not decrease when sample load is lower than 10 micro-g. However, it suddenly decreases when the sample load exceeds 10 micro-g. It is recommended to use the column with the sample load lower than 10 micro-g.

Sample : Lysozyme

Column       : Shodex RSpak RP18-415 (4.6mmID*150mm)
Eluent       : (A);0.1% TFA aq./CH3CN=99/1
               (B);0.1% TFA aq./CH3CN=5/95
               Linear gradient: 0 to 25min : 20%(B) to 60%(B) 
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

RSpak RP18-415 (a column for reversed phase chromatography) can analyze a variety of proteins with good recovery. This column is suitable for fast analysis at high flow rates since the maximum working pressure is 22Mpa.

Recovery of Proteins
1. Ribonuclease A
93%
2. Insulin
98%
3. Cytochrome c
100%
4. Lysozyme
100%
5. BSA
98%
6. Myoglobin
108%
7. Ovalbumin
—
 
Column       : Shodex RSpak RP18-415 (4.6mmID*150mm)
Eluent       : (A); 0.1% TFA aq./CH3CN=99/1
               (B); 0.1% TFA aq./CH3CN=5/95
               Linear gradient: 0 to 25min, 20% (B) to 60% (B)
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Three kinds of protein standards were analyzed using Asahipak C4P-50 4D (a column for reversed phase chromatography).

Sample :
1. BSA
2. Chymotrypsinogen A
3. Ferritin

Column       : Shodex Asahipak C4P-50 4D (4.6mmID*150mm)
Eluent       : (A); 0.1% TFA aq./CH3CN=90/10
               (B); 0.1% TFA aq./CH3CN=5/95
                Linear gradient: 0 to 30min, (A) to (B)
Flow rate    : 0.6mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

Having high resolution, RSpak RP18-415 is suitable for the analysis of complex sample mixtures. Its performance is excellent in determining amino acid sequences in digestive protease of protein.

Sample : Triptic degest of Myoglobin

Column       : Shodex RSpak RP18-415 (4.6mmID*150mm)
Eluent       : (A); 0.1% TFA aq./CH3CN=99/1
               (B); 0.1% TFA aq./CH3CN=5/95
               Linear gradient: 0 to 30min, (A) to 50% (B)
Flow rate    : 0.6mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Asahipak ODP columns are suitable for the analysis of macromolecules such as proteins. A comparison of resolution between the polymer-based (ODP) and the silica-based (ODS) columns shows that the resolution of the former is better.

Sample :
1. Ribonuclease, 2. Cytochrome c, 3. Lysozyme, 4. Chymotrypsinogen A, 5. Ovalbumin

Columns      : Shodex Asahipak ODP-50 6D(6.0mmID*150mm), ODS(6.0mmID*150mm)
Eluent       : 0.05% TFA aq./CH3CN
               Linear gradient: 0 to 30min, 90/10 to 40/60
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

Using gradient elution, a sample containing proteins and peptides with a variety of molecular weight were separated. Asahipak ODP series provides good reproducibility of elution volume as well as high recovery for analysis of proteins and peptides.

No. Proteins
Peptides		 MW VR(mL)
First
Analysis		 VR(mL)
Second
Analysis		 Recovery
(%)
1 Lys-Bradykinin 1188 4.63 4.64 97
2 Bradykinin 1060 5.73 5.70 92
3 Met-Enkephalin 574 7.00 7.00 97
4 Neurotensin 1673 8.04 8.05 99
5 Leu-Enkephalin 556 8.47 8.48 100
6 Substance P 1348 9.24 9.26 93
7 Bacitracin 1450 11.47 11.48 81
8 Insulin 5750 12.24 12.25 95
9 Insulin B chain 3476 12.87 12.89 91
10 Lysozyme 14300 14.43 14.44 96
11 Mastoparan 1479 15.97 15.98 96
12 Myoglobin 17500 18.17 18.18 83 
Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : 0.05% TFA aq./CH3CN
               Linear gradient: 80/20 to 50/50, 20min
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

In analyzing peptides using reversed phase columns (particularly ODS columns), there is always a concern about a decline in recovery. Therefore Asahipak ODP series should be used, which shows a better recovery in analyzing peptides of high, medium and low molecular weights as well as in analyzing acidic, neutral or basic peptides. The recovery of a number of peptides with a wide range of different characteristics are shown for isocratic elution conditions. With these columns it is possible to get a good recovery by isocratic elution.

Substance(1micro-g each) MW pI HP Recovery(%)
Eluent
A		 Eluent
B		 Eluent
C
Glu-Gly-Phe
Substance P Frg.1-4		 Acidic
Basic		 351
497		 3.29
11.44		 2.17
3.24		 96
100		 103
100		 88
110
Met-Enkephalin
Des-Arg9-Bradykinin		 Neutral
Basic		 574
904		 5.90
11.05		 5.02
7.65		 96
–		 96
98		 99
102
Substance P
alpha-Endorphin		 Basic
Neutral		 1348
1684		 12
6.85		 8.61
6.94		 –
–		 90
96		 105
100
beta-Endorphin
Insulin B chain S-sulphonate
Big gastrin (Human)		 Basic
Neutral
Acidic		 3465
3496
3749		 9.93
6.05
4.32		 16.80
24.05
23.53		 –
–
–		 –
–
–		 100
97
95

PI: Isocratic point, HP: Hydrophorbic Parameter, Acidic: Acidic peptide, Neutral: Neutral peptide, Basic: Basic peptide

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : (A); 0.05% TFA aq./CH3CN=85/15
               (B); 0.05% TFA aq./CH3CN=80/20
               (C); 0.05% TFA aq./CH3CN=70/30
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

Basic peptides were analyzed using Asahipak ODP-50 6D (a column for reversed phase chromatography).

Sample :
1. Sub.P Frg.1-4
2. Glu-Gly-Phe
3. Neuro.Frg.8-13
4. Met-Enkephalin
5. alpha-Endorphin
6. Des-Arg9-Bradykinin
7. Substance P

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : 0.05% TFA aq./CH3CN=80/20
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

A sample of different proteins and peptides was separated with a various pH values of the eluent. Under acidic conditions (pH2.3, pH3.0), Leu-Enkephalin and Leu-Trp-Met have been strongly retained and the elution times for the substances are quite similar; therefore, the separation is not satisfactory. In contrast, when an alkaline according to the fact that, under alkaline condition (pH10) with Asahipak ODP column, a better separation with sharper peaks of the substances has been obtained in a shorter time. In addition, ODP columns have higher theoretical plate numbers under alkaline conditions.
Refer to Analysis of Oligopeptides with Acidic and Alkaline Eluent.

Sample :
1. Glu-Gly-Phe
2. Met-Enkephalin
3. Leu-Enkephalin
4. Leu-Trp-Met

Theoretical plate number

No.
pH2.3
pH3
pH6.7
pH10
1
 6100 1100 3800 –
2
 7400 1100 4700 5700
3
 7600 600 3800 6700
4
 6100 500 2400 8000 
Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : buffer/CH3CN=85/15
               (pH2.3)  0.05% TFA aq.
               (pH3.0)  50mM Acetic acid aq.
               (pH6.7)  50mM Ammonium acetare buffer
               (pH10.0) 50mM Ammonium acetare buffer
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

Oligopeptides were analyzed using Asahipak ODP-50 6D (a column for reversed phase chromatography) with acidic and alkaline eluent.
Please refer to Effect of Eluent pH (ODP).

Sample :
1. Glu-Gly-Phe
2. Met-Enkephalin
3. Leu-Enkephalin
4. Leu-Trp-Met

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : (1); 0.05% TFA aq./CH3CN=85/15
               (2); 50mM Ammonium acetate(pH10.0)/CH3CN=85/15
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

The difference of retention of three columns for reversed phase chromatography, Asahipak ODP, C8P and C4P were compared.

Sample :
1. beta-Endorphin
2. Big gastrin
3. Insulin

Columns      : Shodex Asahipak ODP-50, C8P-50, C4P-50 (4.6mmID*150mm each)
Eluent       : 0.05% TFA aq./CH3CN=72/28
Flow rate    : 0.6mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

Compared to Asahipak ODP columns, C8P columns allow the analysis of proteins and peptides under more moderate conditions because the organic solvent concentration in the eluent can be lowered.

Sample :
1. Substance P
2. beta-Endorphin
3. Insulin B chain
4. Insulin

Column       : Shodex Asahipak C8P-50 4D (4.6mmID*150mm)
Eluent       : 0.05% TFA aq./CH3CN=73/27
Flow rate    : 0.6mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

Oligopeptides were analyzed using Asahipak ODP-50 6D (a column for reversed phase chromatography).

Sample :
1. Val-Glu-Glu-Ala-Glu
2. Gly-Gly-Phe
3. Met-Enkephalin
4. Bradykinin
5. Angiotensin II
6. Angiotensin III
7. Phe-Phe-Phe

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : (1): (A); 0.1M NaClO4 + 0.1% Sodium phosphate(pH2)
                    (B); CH3CN
               Linear gradient: 0min to 60min, (A)/(B)=90/10 to 60/40
               (2): (A); 0.1M NaClO4 + 0.1% Sodium phosphate(pH9)
                    (B); CH3CN
               Linear gradient: 0min to 60min, (A)/(B)=90/10 to 60/40
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

Oligopeptides were analyzed using Asahipak ODP-50 6D (a column for reversed phase chromatography).

Sample :
1. Gly-Gly-Ala
2. Gly-Gly-Leu
3. Leu-Enkephalin
4. Met-Enkephalin

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : 0.05% TFA aq./CH3CN=80/20
Flow rate    : 1.0mL/min
Detector     : UV(210nm)
Column temp. : 30deg-C

Dipeptides were analyzed using RSpak DE-613 (a column for reversed phase chromatography).

Sample :
1. Gly-Val
2. Phe-Gly
3. Ala-Phe
4. Val-Phe
5. Tyr-Leu

Column       : Shodex RSpak DE-613 (6.0mmID*150mm)
Eluent       : (A); 0.05% TFA aq./CH3CN=96/4
               (B); 0.05% TFA aq./CH3CN=75/25
               Linear gradient: 0 to 30min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

Angiotensins were analyzed using Asahipak ODP-50 6D (a column for reversed phase chromatography).

Sample :
1. Val5-Angiotensin II
2. Angiotensin II
3. Angiotensin III
4. Asn1,Val5-Angiotensin I
5. Val5-Angiotensin I
6. Angiotensin I

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : 0.05% TFA aq./CH3CN=80/20
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

Opioid peptides were analyzed using Asahipak ODP-50 6D (a column for reversed phase chromatography) by post-column reaction method.

Sample :
1. Tyr-Gly
2. Tyr-Gly-Gly
3. Tyr-Gly-Gly-Phe
4. Tyr-Phe
5. ME-Thr-ser-Glu-Lys
6. ME, Met-Enkephalin
7. LE, Leu-Enkephalin
8. ME-Arg, Met-Enkephalin-Arg
9. alpha-EDP, alpha-Endorphin
10. LE-Arg, Leu-Enkephalin-Arg
11. ME-NH2, Met-Enkephalin-NH2
12. gamma-EDP, alpha-EDP-Leu
13. ME-Arg-Glu-Leu
14. LE-NH2, Leu-Enkephalin-NH2
15. ME-Arg-Phe
16. DNP 1-8

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : (A); 50mM Sodium borate(pH10.0)/H2O/CH3CN=4/15/1
               (B); 50mM Sodium borate(pH10.0)/H2O/CH3CN=1/1/3
               Gradient: (A) to (B)
Flow rate    : 1.0mL/min
Detector     : Fluorescnce(Ex. 330nm, Em. 430nm)
Column temp. : Room temp.

Guo-Qing Zhang et al., Analytical Science, 6 (1990) 671.

Opioid peptides were analyzed using Asahipak ODP-50 6D by pre-column reaction method.

Sample :
1. Tyr-Gly-Gly-Phe
2. ME, Met-Enkephalin
3. LE, Leu-Enkephalin
4. LE-Arg
5. alpha-EDP, alpha-Endorphin
6. LE-NH2
7. DNP 1-7
8. [D-Ala2] LE-NH2
9. ME-Arg-Gly-Leu
10. gamma-EDP
11. ME-Arg-Phe
12. DNP 1-8
13. beta-NED, beta-Neoendorphin
14. alpha-NED, beta-NED-Lys
15. DNP 1-9
16. DNP 1-10
17. DNP-B

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : (A); 0.1M Borate buffer(pH9.5)/H2O/CH3CN=10/39/1
               (B); 0.1M Borate buffer(pH9.5)/H2O/CH3CN=2/3/5
               Gradient:(A) to (B)
Flow rate    : (0 to 25min); 0.8mL/min, (25 to 55min); 1.0mL/min, (after 55min); 0.8mL/min 
Detector     : Fluorescence(Ex. 330nm, Em. 430nm)
Column temp. : Room temp.

Guo-Qing Zhang et al., Chem. Pharm. Bull., 39 (1991) 126.

Tryptic digest of myoglobin were analyzed using Asahipak ODP-50 6D (a column for reversed phase chromatography).

Abbreviation of Amino Acids

Abbreviation of Amino Acid
A Ala M Met
C Cys N Asn
D Asp P Pro
E Glu Q Gln
F Phe R Arg
G Gly S Ser
H His T Thr
I Ile V Val
K Lys W Trp
L Leu Y Tyr

Sample : Tryptic digest of Myoglobin

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : (A); 0.07% TFA aq.
               (B); CH3CN containing 0.05% TFA
               Initial ratio (A)/(B)=90/10
               Linear gradient of 1%/min
Flow rate    : 0.8mL/min
Detector     : UV(214nm)
Column temp. : 30deg-C

Coutesy of Dr. Takano, Institute for Protein Research, Osaka University

Whey proteins were analyzed using RSpak RP18-415 (a column for reversed phase chromatography).

Sample: Whey protein
1. gamma-Lactoalbumin
2. beta-Lactoglobulin A
3. beta-Lactoglobulin B

Column       : Shodex RSpak RP18-415 (4.6mmID*150mm)
Eluent       : (A); 0.1% TFA aq.
               (B); 0.1% TFA in CH3CN
               Linear gradient: 0 to 30min, 30% (B) to 50% (B)
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Reduced glutathione (GSH) and oxidized glutathione (GSSG) are separated with using polymer based reversed phase column, Asahipak ODP-50 6E.

Sample : 5ppm each,
1. Glutathione, GSH (MW: 307.33)
2. Glutathione disulfide, GSSG (MW: 612.63)

Column       : Shodex Asahipak ODP-50 6E (6.0mmID*250mm)
Eluent       : 50mM Sodium phosphate buffer(pH2.5)
Reagent      : 50mM o-Phthalaldehyde in CH3OH/200mM Sodium phoshate buffer(pH12.0)/2-Mercaptoethanol
               =50/450/1
Flow rate    : (eluent); 0.5mL/min, (reagent); 0.3mL/min
Detector     : Fluorescnce(Ex. 340nm, Em. 420nm)
Column temp. : 40deg-C
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