High efficiency RP-HPLC separation of peptides and proteins using high-temperature

S120515AE

Comparison of separation of peptides and proteins between at 40℃ and 70℃
Condition A (MW 500-18,400)
Peak capacity= 230mAU
60

21.8-25.8 MPa(3,160-3,740 psi)

50

40ºCCondition B (MW 14,300-25,700)

160

37.0-37.5 MPa(5,370-5,440 psi)

50

40

14, 3

1. Oxytocin
2. Leu-Enkephalin

33. β-Endorphin

30

52020

10

1000F111228B

0.00.51.0

1.5

2.0mAU

2.5

160

15.8-18.5 MPa(2,290-2,680 psi)

50

3.0

3.5

min*

0.0

60

0.5

1.0

1.5

2.0

2.5

40

3.0

3.5

3 4

320

10

1000F111228A

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

min*

F111226A

0.0

40℃

70℃

1,007

0.017

0.014

556

0.015

0.015

3,465

-

0.016

4. Insulin

5,733

-

0.015

5. β-Lactoglobulin A

18,400

0.043

0.030

1. Lysozyme

14,300

0.069

0.044

2. α-Chymotrypsinogen25,700

0.080

0.049

3. β-Lactoglobulin A

18,400

0.080

0.048

ColumnEluent30

520min*240304.0

Peak capacity= 107

127.4-28.4 MPa(3,970-4,120 psi)

50

Peak
Peak width
width ½½(min)(min)Condition B

F111226BmAU

Peak capacity
2 = 279

MWMWCondition A

2403070ºC

Peak capacity= 67mAU

2Analytes

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0min*: YMC-Triart C18 (1.9 µm, 12 nm), 50 X 2.0 mmI.D.
: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1) - condition AB) acetonitrile/IPA/TFA (50/50/0.1) - condition B
Gradient : 10-80%B (0-5 min) - condition A
30-60%B (0-5 min) - condition B
Flow rate : 0.4 mL/min
Detection : UV at 220 nm
Injection : 1 µL (50 µg/mL) - condition A
1 µL (250 µg/mL) - condition B
System : Agilent 1200SL

The separation of peptides and proteins with a variety of molecular weight (MW) is compared increasing column temperature from 40℃ to70℃.
Although adding stronger solvent like IPA to acetonitrile of the mobile phase (condition B) is effective to reduce larger protein retention andimprove peak shape, the molecules with MW >10,000 still result in peak broadening at 40℃, as shown in the upper chromatograms.
Increasing column temperature to 70℃ provides selectivity change, sharper peaks, and therefore, improved resolution especially for larger
molecules. Generally, larger molecules diffuse very slowly compared to small molecules. An elevated temperature can improve efficiency
and peak shape by lowering mobile phase viscosity and improving mass transfer, and the appropriate MW range for pore size of packing
materials can be more expanded than that at a lower temperature.
Temperature is a simple and effective tool to increase resolution in separation of proteins and peptides.

Improvement of resolution by increasing column temperature and coupling of 1.9 µm columns
40ºC

Peak capacity = 365mAU

1.9 µm, 100 X 2.0 mmI.D.
30
15 min gradient46.5-48.5 MPa (6,740-7,030 psi)

20
100070ºC

246810P110705A
1415 min12Peak capacity = 450

23% more peaks can be resolved by increasing thecolumn temperature to 70℃ in the separation oftryptic digest of Hemoglobin.

25mAUmAU

1.9 µm, 100 X 2.0 mmI.D. 30
15 min gradient27.6-28.6 MPa (4,000-4,150 psi)

2010900mAU

Two coupled30
1.9 µm, 100 X 2.0 mmI.D.
2030 min gradient58.1-61.6 MPa (8,430-8,930 psi)

9.4

9.8

10.2 10.6 min

P110728A
246810121415 min

25mAU

Peak capacity = 630

101902021min

P110707A
048121620242830 min

The outstanding efficiency obtained by a coupling of
two 100 mm length of Triart 1.9 µm columns
reduces co-elution peaks and allows the precise
separation in an analysis of complicated samples,such as peptide mapping.
Column : YMC-Triart C18 (1.9 µm, 12 nm): A) water/TFA (100/0.1)
EluentB) acetonitrile/TFA (100/0.08)
5-40%B (0-15 min) for a single column
5-40%B (0-30 min) for two coupled columns
Flow rate : 0.4 mL/min
Detection : UV at 220 nm
Injection : 10 µL for a single column
20 µL for two coupled columns
Sample : Tryptic digest of Bovine Hemoglobin
System : Agilent 1290

Comparison of retention stability of RP columns at pH 1 (1% TFA) and 70℃% of initial retention time

120%
100%

YMC-Triart C18
YMC-Triart C880%

Silica based C1860%

Hybrid-silica based
C1840%
20%0%0204060
Time (hr)80100Test condition
The columns are kept in acetonitrile/water/TFA (10/90/1, pH 1) at 70oC, and tested
for performance every 20 hours under thecondition of column inspection below.
[Column inspection]
Column: 5 µm, 50 X 2.0 mmI.D.
Eluent: acetonitrile/water (60/40)
Flow rate: 0.2 mL/min
Temperature : 37oC
: butyl benzoate
SampleNewly developed hybrid particles and surface
modification of Triart C18 and C8 provide excellent
durability in the difficult conditions such as strongly
acidic at elevated temperature (1% TFA, 70℃).This
advantage enables a rapid and efficient method
development of a complex mixture of peptides andproteins.