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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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