Particle size and pore size of silica are very important
parameters of a reversed phase column for HPLC
analysis. Small particle sizes, less than 3 µm and
including sub-2µm, have been widely used for achieving
high performance and fast separations. One additional
factor that should be considered is the pore size of the
silica particle. Normally you use column packings with
small pores (60-120Å) for small molecules (less than
5000 Da), use 150-300Å for peptides and low molecular
weight proteins and use large pore sizes (1000Å-4000Å)
for very high molecular weight proteins and vaccines.
A small protein, such as insulin with a molecular
weight of about 5800 Da could potentially be analyzed
on columns with small pores or slightly larger pores. To
maximize efficiency smaller particle sizes with an
optimum pore size should be selected. An insulin
regulatory method (USP) with an isocratic mobile
phase was used in this study of optimum pore and
particle size. The performance and resolution is
compared among columns with different pore size
including 80Å, 95Å, 120Å, 170Å and 300Å and as well as
different particle sizes including 1.8µm, 2.7µm, 3.5µm
and 5µm. These choices also include different particle
types, including both superficially porous and totally
porous particles. The end result of the work shown
here are recommendations for achieving the highest
efficiency and resolution while still meeting the
requirements of the regulatory methods.
• Columns with a small a pore size, <100Å did not
provide the best results for insulin, because of the
restricted access to the bonded phase in the pores of
these columns.
• Columns with a larger pore size, >100Å, such as the
Poroshell 120, Agilent TC-C18(2), and 300SB-C18
provided much higher efficiency and lower tailing
factors. A pore size as large as 300Å was not needed
for an efficient separation of insulin. The intermediate
pore size columns, the Poroshell 120 and TC-C18 (2)
columns were suitable and would therefore be a good
choice for separations of other small proteins or peptide
mapping.
• The smaller particle sizes provided the highest
efficiencies. The Poroshell 120 columns had a 2.7µm
particle size with a pore size of 120Å and were suitable
for the highly efficient analysis of insulin.
[1] China Pharmacopoeia (2010 edition), insulin, page. 845 – 846. [2] The United States Pharmacopoeia USP 31 (vol 2) – Insulin, page. 2403 – 2404. [3] Phu T Duong, Analysis of Oxidized Insulin chains using Reversed Phase Agilent ZORBAX RRHD 300 SB-C18, Agilent application note, 5990-7988EN To access Agilent’s Literature Library for application notes, visit us online at www.agilent.com/chem
Results and Discussion Conclusions
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Pittcon 2012
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March 13, 2012
1100-4P
The influence of silica pore size and particle size on insulin - a small molecule of protein separation Rongjie FU1, William LONG2, Anne MACK2 and Maureen JOSEPH2 1Agilent Technologies (Shanghai) Co., Ltd, Shanghai, China 200131 2Agilent Technologies, Inc. 2850 Centerville Rd. Wilmington, Del. USA 19808
Experimental Abstract
All the columns explored in this paper were C18
columns, meeting pharmacopeia definitions for
octadecyl silane (C18), chemically bonded to porous
silica.
HPLC Conditions
Sample: Porcine insulin (NIFDC China)
Columns: Agilent ZORBAX SB-C18, 4.6 x 150 mm, 5 µm
(p/n 883975-902)
ZORBAX Eclipse Plus C18, 4.6 x 150 mm, 5 µm
(p/n 959993-902)
ZORBAX 300SB-C18, 4.6 x 150 mm, 5 µm
(p/n 883995-902)
ZORBAX SB-C18, 4.6 x 100 mm, 3.5 µm
(p/n 861953-902)
ZORBAX Eclipse Plus C18, 4.6 x 100 mm, 3.5 µm
(p/n 959961-902)
ZORBAX SB-C18, 4.6 x 100 mm, 1.8 µm
(p/n 828975-902)
ZORBAX Eclipse Plus C18, 4.6 x 100 mm, 1.8 µm
(p/n 959964-902)
Agilent Poroshell 120 SB-C18, 4.6 x 100 mm, 2.7 µm
(p/n 685975-902)
Agilent Poroshell 120 EC-C18, 4.6 x 100 mm, 2.7 µm
(p/n 695975-902)
Agilent TC-C18(2), 4.6 x 150 mm, 5 µm
(p/n 588935-902)
System: Agilent 1200 SL LC with binary pump,
thermostatted column compartment, high performance
autosampler and diode array detector
Mobile Phase: 74% A:26% B, where A: 0.2 mol/L sulfate
(dissolve 28.4g anhydrous sodium sulfate in 1000 mL of
water, pipet 2.7 mL of phosphoric acid into the solution
and adjust with ethanolamine to pH 2.3, and mix) B:
acetonitrile
Flow Rate: 1.0 mL/min
Injection Vol: 20 µL
Temp: 40 °C
Detector: UV 214 nm
Reference
The method for insulin was first run on four traditional
columns with a 5 μm particle size, but with four different
pore sizes.
Insulin has the poorest performance both on the SB-C18, 5
μm and the Eclipse Plus C18, 5 μm columns with the smallest
pore sizes of 80Å and 95Å. The Agilent ZORBAX 300SB-C18
column has the lowest surface area, but the retention is only
slightly less than the small pore size columns. Once the
insulin can access the pores and interact with all of the
bonded phase in the pores it is better retained. This more
efficient access is also seen in the improved peak shape,
even on a non-endcapped bonded phase. The Agilent TC-
C18(2) column has the highest surface area and the
strongest retention, most likely due to accessing all the
pores and retention increasing with this higher surface area
of silica particles (290 m2/g).
In these chromatograms the Poroshell 120 SB-C18 2.7um column
provides 2X the efficiency of the 80Å SB-C18 1.8µm. This is due to
the larger pore size and more rapid diffusion in the 120Å pores.
Switching from the ZORBAX Eclipse Plus C18 to Poroshell 120 EC-
C18 also provided an increase in efficiency with the change from
95Å to 120Å pore size. In addition, the peak shape of insulin on
both Poroshell 120 columns was improved with greater access to
the pores.
In addition, the Poroshell 120 column provides higher efficiency
than the 5um Agilent TC-C18 (2). The chromatogram shown on
the TC-C18 (2) column is the one run with 27.5% organic in the
mobile phase so that the retention times could be more closely
matched. The improvement in the efficiency when comparing
these two columns is due to the benefit of the smaller particle
size with a larger pore size.
The retention on the Agilent TC-C18(2) column is very long, at
over 40 minutes, and not ideal. Therefore the retention is
reduced from 44 minutes to 15 minutes by increasing the
organic in the mobile phase by only 1.5% while still
maintaining good peak shape and efficiency
The insulin method was run on 5 μm, 3.5 μm, and 1.8 μm
Agilent ZORBAX SB-C18 columns and Agilent ZORBAX Eclipse
Plus C18 columns. The data demonstrate improved efficiency
using the smaller 1.8 μm particle columns. While these
columns are not the ideal pore size columns, efficiency should
still be expected to improve with smaller particle sizes, as it
does here with insulin.
Results and Discussion Results and Discussion
Influence of silica pore size, 5µm
particles
min 10 20 30 40 50
mAU
0
25
50
75
100
125
150
min 10 20 30 40 50
min 10 20 30 40 50
mAU
0
50
100
150
200
250
min 10 20 30 40 50
mAU
0
50
100
150
200
250
mAU
0
100
200
300
400
500
80Å Agilent ZORBAX SB-C18
4.6X150mm, 5um
1
2
1
2
1
2
1
2
N 1 = 1776 Tf 1 = 1.7 N 2 = 2774
Tf 2 = 2.0
Rs=3.4
1. Porcine insulin 2. A - 21 desamido insulin
N 1 = 1845 Tf 1 = 1.2
N 2 = 2489 Tf 2 = 1.2
Rs=3.1
N 1 = 5124 Tf 1 = 1.1 N 2 = 5870
Tf 2 = 1.0
Rs=4.5
N 1 = 4333 Tf 1 = 1.1
N 2 = 4709 Tf 2 = 1.0
Rs=3.9
95Å Agilent ZORBAX Eclipse Plus C18
4.6X150mm, 5um
170Å Agilent TC-C18(2) 4.6X150mm, 5um
300Å Agilent ZORBAX 300SB-C18 - ,
4.6X150mm, 5um
min 10 20 30 40 50 60
mAU
- 25
0
25
50
75
100
125
150
175
min 10 20 30 40 50 60
mAU
0
100
200
300
400
500
N 1 = 5124 Tf 1 = 1.1
N 2 = 5870 Tf 2 = 1.0
Rs=4.5
1 1 Porcine insulin 2 A-21 desamido insulin
2
N 1 = 4527 Tf 1 = 1.0
N 2 = 4904 Tf 2 = 1.0
Rs=3.4
1
2
26%ACN
27.5%ACN
Influence of silica particle size
Agilent ZORBAX SB-C18
4.6x150mm, 5 um
4.6x100mm, 1.8 um
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
50
100
150
200
250
300
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
500
4.6x100mm, 3.5 um
1
2
1
2
1
2
N1= 1776
Tf1= 1.7 N2= 2774 Tf2= 2.0
Rs=3.4
N1= 1806
Tf1= 1.2
Rs=2.8
Rs=4.9
1. Porcine insulin
Agilent ZORBAX Plus C18
4.6x150mm, 5um
Agilent ZORBAX Plus C18,
4.6x100mm, 3.5 um
Agilent ZORBAX Plus C18,
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
50
100
150
200
250
300
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
500
600
1
2
1
2
1
2
Rs=6.0
Rs=3.0
Rs=3.1
1. Porcine insulin
Comparison between superficially porous
and totally porous columns
N 1 = 5215 Tf 1 =1.4 N 2 = 6691
N 2 = 1.2
Agilent ZORBAX Eclipse Plus C18 4.6x100mm, 1.8um
Agilent Poroshell 120 SB-C18 4.6x100mm, 2.7um
Agilent ZORBAX SB-C18
4.6x100mm, 1.8um
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
500
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0 100 200 300 400 500 600
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0 100 200 300 400 500 600
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0 100 200 300 400 500 600
N 1 = 10860 Tf 1 = 1.1 N 2 = 12377
Tf 2 = 1.0
N1=7854
Tf1=1.4 N2= 9434 Tf2= 1.1
N1= 9768
Tf1= 1.0 N2= 10710 Tf2= 1.0
1 Porcine insulin 2 A21 desamido insulin
255 bar
190 bar
237 bar
198 bar
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
500
600
700
min 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
500
600
700
N1=10860
Tf1= 1.1
N2 = 12377
Tf2 = 1.0
Poroshell 120 SB-C18
4.6x100mm, 2.7 um Rs=6.6
N1= 4527
Tf1= 1.0
N2= 4904 Tf2= 1.0
Rs=3.4
1
2
Agilent TC-C18(2) 4.6x150mm, 5 um
1
2
27.5%ACN
26%ACN
1 Porcine insulin
Agilent Poroshell 120 EC-C18
4.6x100mm, 2.7um
2 A21 desamido insulin
2. A21 desamido insulin
Agilent ZORBAX SB-C18
N2= 2172
Tf2= 1.2
Agilent ZORBAX SB-C18
Tf2= 1.2
N2= 6691 N1= 5215
Tf1= 1.4
4.6x100mm, 1.8 um
2 A21 desamido insulin N1= 1845
Tf1= 1.2
N1= 2157 Tf1= 1.2
N1= 7854
Tf1= 1.4
Tf2= 1.2
N2= 2489
Tf2= 1.2
N2= 2604
Tf2= 1.1
N2= 9434
Agilent TC-C18(2)
4.6X150mm, 5um
Agilent TC-C18(2)
4.6X150mm, 5um
