Some Hint on How to Make a Standard UHPLC Column
with + 300 000 Theoretical Plates/MeterNorikazu Nagae1, Tomoyasu Tsukamoto1, Henrik Svennberg2
1 ChromaNik Technologies Inc. Namiyoke, Minato-ku, Osaka Japanwww.chromanik.co.jp

2 Biotech AB, Innovation, Onsala, Sweden

The race to achieve the highest number of theoretical plate counts has been going for many years already, even though it could and should be argued that agood separation is mainly governed by good selectivity, a reasonable retention and then high column efficiency. However, the race has still been going, and thispresentation will show how + 300 000 theoretical plates/meter can be achieved in a standard column for UHPLC. The importance of a good bonding and end capping
technique will be discussed and hints about how this is made is revealed. The importance of base particle choice with a good heat transfer capacity will be shown and theimportance of small extra column peak broadening will be emphasized. The + 300 000 theoretical plates/meter is shown in two different columns lengths with the innerdiameter of 2.1 mm, 150 and 50 mm long respectively.

Effect of end-capping

Effect of frictional heating

End-capping with hexamethyltrisiloxane and TMS on C18silica

HOSiOSiSiOSiOOOSiSiSiSiOSiOOSiOOO OOOO O Si
SiSiOO OHOOSiOOSiSiSi
OO O
O O
OOSi
Si OOSiOOSi

O O
SiOOOO

Connecting tube usedoo

22.8 C26.5 C302520150246810

Measurement condition
400.5

11.5

30000

25000

N3=303,060/m
4100.5

11.5

Totally porous hybrid C18 1.7 μm

SunShell C18 2 μm00.1

Stationary phase

0.2

0.3

0.4

0.5

0.6

0.7

Regarding a totally porous hybrid
silica particle, ethylene chain in
the skeletal structure of silica leads
thermal conductivity to reduce.
Consequently, biased heat
distribution makes plate numberlow.

*Fabrice Gritti, Georges Guiochon, J. Chromatogr. A 1217 (2010) 5069.
Column dimension, 100 x 2.1 mm; mobilephase, CH3CN/H2O=60/40; temperature, 40
A part of abstractºC; sample, acenaphthene; UHPLCThis unexpected result is accounted for by the three times smaller
heat conductivity of the BEH bed (BEH 0.25 W/m/K) than that ofinstrument, X-LC with 0.1 mm i.d.
the Kinetex bed (Kinetex 0.75 W/m/K).
connecting tube (JASCO).

C18

2Both Sunniest end-capping and Silanol Activity Control
C18C180

1

2

33

45X-LC with MarvelX

32SunShell C18 2 μm

34Company P
Core Shell C18
1.7 μm1

Carbon loading and retention factor (reaction at two different temperatures)1o(X-40) C(more than 200 oC )

7.0%

7.0%

7.3%

7.7%

(cut off some C18chains by heat)(not cut off C18
chain by heat)

Silanol activity control

Yes

NoRetention factor (k)

10.6

9.5

*Base material: Core shell silica, 2.6 μm

X oCo10% reduce0246810121416(X-40) C182022X oC was too high to keep
carbon loading of initial
C18. (X-40) oC could keep
carbon loading. As a result,carbon loading after endcapping at (X-40) oC washigher than that at X oC.

217.3

125

9.7

8.0

133

PoroShell C18 EC, 2.7 μm

9.0

8.5

135

Cortecs C18, 2.7 μm

7.77.3113

Accucore C18, 2.6 μm

7.4

8.8

130

Kinetex C18, 2.6 μm

5.4

4.9

102

a. Retention factor of amylbenzene, mobile phase; methanol:water=75:25, 40 oC.
b. Measured after C18 packing material was pulled out of a column.
c. Measured using C18 materials sintered at 600 degree Celsius for 8 hours.

40

It is considered that lessresidual silanol groupmade hydrophobicity ofa stationary phase high,then made retention
factor large.

123

45678943.9605

N4=5,300, TF4=2.73

Company T C18 2.6 μmN4=4,800, TF4=3.12

Company W C18 2.7 μm

N4=3,400, TF4=3.24

Company S C18 2.7 μm

10CH3N
CH3Amitriptyline

SunShell C18 showed the highest theoretical plate (N) and the lowest tailingfactor. Highly-effective end-capping including heat treatment (silanol activitycontrol) reduced tailing for a basic compound. As a consequence, highefficiency for a reversed column was achieved by highly-effective end-capping.

456SunShell C18 2 μm, 150 x 2.1 mm
X-LC with SUS

N4=307,300/m

24

N4=46,100

Measurement conditionColumn, SunShell C18, 2 μm 150 x 2.1mm; mobile phase, CH3CN/H2O=70/30;
flow rate, 0.4 mL/min; temperature, 25oC; pressure, 64 MPa; detection,UV@250 nm; injection volume, 0.4 μL;
sample, 1=uracil, 2=ethylbenzene,
3=acenaphthene, 4=butylbenzene.

Company PCore Shell C18 1.7 μm
Company W totally porous
hybrid C18 1.7 μm
3Company S totally porousmonodisperse C18 1.9 μm

2123456Retention time/min

Column dimension, 100 x 2.1 mm; mobile phase, methanol/water = 30/70; flow rate, 0.2 mL/min;temperature: 40 oC; sample, 1=uracil, 2=pyridine, 3=phenol; UHPLC instrument, X-LC with 0.1 mm i.d.
connecting tube (JASCO).
43121243432101143223324623456Company S
Core Shell C18 2 μm

2101SunShell C18 2 μm

10Retention time/min

3322Company P C18 2.6 μm

2Retention time/min

1

N4=5,200, TF4=2.61

Column dimension, 150 x 4.6 mm; mobile phase, acetonitrile/10mM
ammonium acetate pH6.8 = 40/60; flow rate, 1.0 mL/min; temp.: 40oC;
sample, 1=uracil, 2=propranolol, 3=nortriptyline, 4=amitriptyline.

1.05

1SUS
Marvel X
41900
48912 17% up
42934
44037 2.7% up
38989
40899 4.9% up
38125
39947 4.8% up
1.176
1.207
1.015
1.024
1.001
1.020
1.048
1.090
0.0074 0.0070
0.0172 0.0170
0.0395 0.0390
0.0542 0.0533

Retention time/min

04
4

26,59203

N4=12,800, TF4=1.89 Company A C18 2.7 μm
4269

21SunShell C18 showed thelargest retention factor
although carbon loading
was not the highest.

N4=16,700, TF4=1.20 SunShell C18 2.6 μm
454.1

343

1.01

3Both Sunniest end-capping and Silanol Activity Control

2458

14,5111Comparison of amitriptyline peak
1477

53.8

3

110.4

53.5

0.97

2

Relationship between retention factor and carbon loading

Ascentis Express C18, 2.7 μm

1.1924,700124SunShell C18, 2.6 μm

25,533

Peak No.
1234
12Tailing factor
34
1
Peak width,
2h0.5 (min)34Efficiency

Peak shape and efficiency of pyridine and amitriptylineRegarding retention factor,reversed result wasobtained. Less silanol groupmade hydrophobicity ofstationary phase high.

CarbonSpecific surface
loading b (%) area b, c (m2/g)

Theoreticalplate /pressure(MPa-1)
613

Company W totally
porous hybrid C181.7 μm

4Retention time/min

Retention
factor a48.5

Pressure(MPa)Column, SunShell C18 2 μm 100 x 2.1 mm, company S
core shell C18 2 μm 100 x 2.1 mm, company P core shell
C18 1.7 μm 100 x 2.1 mm, company W C18 1.7 μm 100 xCompany S totally porous 2.1 mm, company S monodisperse C18 1.9 μm 100 x 2.1
3monodisperse C18 1.9 μm mm; mobile phase, CH3CN/H2O=60/40; flow rate, 0.54mL/min; temperature, 40 ºC; sample, 1=uracil,
2=ethylbenzoate, 3=acenaphthene, 4=butylbenzene;
UHPLC instrument, X-LC with 0.1 mm i.d. connecting
Retention time/min
tube (JASCO).

32X oC

1.04

Theoretical plate Tailing factor

SunShell C18
Company S
Core Shell C18
Company P
Core Shell C18
Company W totally
porous hybrid C18
Company S totally
porous monodisperseC18

Company SCore Shell C18 2 μm41264129,721

Acenaphthene
41More hydrophobicEnd-capping layer

Carbon loading
after end-capping

Column, SunShell C18, 2.6 μm 150 x 4.6 mm; mobilephase, CH3CN/H2O=70/40; flow rate, 1.8 mL/min;temperature, RT; detection, UV@250 nm; injection
volume, 0.4 μL; sample, 1=uracil, 2=ethylbenzene,
3=acenaphthene, 4=butylbenzene.

N1=326,080/m

32C18Carbon loading of only C18

Measurement condition

412End-capping reaction
temperature

2SunShell C18 2.6 μm, 150 x 4.6 mm[[An extremely low dead volume column hardware manufactured by Tomoe Works was used.]]
3X-LC with SUS

Efficiency and back pressure

Stationary phase

SUS
Marvel X
1208
3593 197% up
7720
12625 64% up
13589
15153 12% up
13936
14733
6% up
2.326
1.445
1.401
1.286
1.048
1.006
0.997
0.972
0.0094 0.0051
0.0107 0.0083
0.0201 0.0194
0.0297 0.0295

Efficiency loss by generation of frictional heating

15000

Flow rate, ｍL/min

C18

Peak No.
1234
12
Tailing factor
34
12Peak width,h0.5 (min)34
EfficiencyRetention time/min

20000

Totally porous monodisperse C18 1.9 μm

End-capping layer

C18232A core shell silica particle
has enough high thermal
conductivity to makefrictional heating diffuse.
Heat distribution is madeuniform in the column.

5000

C18Column, SunShell C18, 2 μm 50 x 2.1 mm; mobilephase, CH3CN/H2O=60/40; flow rate, 0.6 mL/min;temperature, RT; detection, UV@250 nm; injection
volume, 0.4 μL; sample, 1=uracil, 2=ethylbenzene,
3=acenaphthene, 4=butylbenzene.

2Temperature rose 14 oC at the outlet of thecolumn by effect of frictional heating.

1235000

Comparison of 2 kinds of end-capping

C183.93 μL
2.21 μL

MarvelX

31

Efficiency loss by thermal friction*

10000

Conventional end-capping

Tube volumeSunShell C18 2 μm, 50 x 2.1 mm

X-LC with MarvelXO H Hydrated silanol group without influence of alkyl groups

C18PeekSil 0.1 mm i.d. x 200 mm

100O H Non hydrated silanol group by influence of hydrophobicity of alkyl groups

C1837.0 C

35.3 C31.2 C

5Plate numberHeatingColumn to flow cell

SUS 0.1 mm i.d. x 300 mm

X-LC with SUS

A basic compoundshows no tailing on
Sunrise C18-SAC
because hydrated
silanol groups don’t
make a basic
compound tailing aswell as silica column
on HILIC mode showsno tailing for a basiccompound.

Change overto siloxane
bondInjector to column

X-LC with SUS

Column, core shell C18 2.6 μm, 150 x 4.6 mm;
mobile phase, methanol; flow rate, 5 mL/min;column back pressure, 70 MPa; temperature, 23 oC(room temperature).

35

Elution time, min

Silanol groups
with highabsorption activity

ooo40

Sunrise C18-SAC

Silanol Activity
ControlTechnology

Instrument*X-LC, is a UHPLC instrument manufactured by Jasco. *MarvelX is a connecting tube manufactured by IDEX.

Measurement point

Another end-capping with heating on C18 silica, reduce ofsilanol groups
Typical no end-capping C18

Comparison of an inner diameter of tubing in the UHPLC
instrument

After 5.5 min

3OO

After 3 min

After 1.5 min

X-LC with MarvelX MarvelX 0.075 mm i.d. x 350 mm MarvelX 0.075 mm i.d. x 150 mm

We named this endcapping method
Sunniest end-capping.

SiOOOOO

Si OSiHOSiOSi O OOOO

SiSiSiSiSiSi OO OOSiSiO

After 0.5 min
Flow

Temperature, ℃

End-capping
reagent

Frictional heating phenomenon
Before a flow

End-capping reagent
moves like a Geometrid
caterpillar, so that afunctional group on thetip of the arm can bondwith a silanol groupwhich Is locatedanywhere.

Final TMS

C18

Effect of extra-column volume

4

10

SunShell C18 2 μm

N4=6,778, TF4=3.58

Company S
Core Shell C18 2 μm

N4=7,059, TF4=4.39

Company PCore Shell C18 1.7 μm

N4=14,555, TF4=1.27

Company W totally porous
hybrid C18 1.7 μm

5Retention time/min

8N4=21,096, TF4=1.18

12Retention time/min

64 N4=9,371, TF4=4.72

14Company S totally porousmonodisperse C18 1.9 μm

16Column dimension, 100 x 2.1 mm; mobile phase, acetonitrile/10mM ammonium acetate pH6.8 = 40/60;
flow rate, 0.3 mL/min; temperature, 40oC; sample, 1=uracil, 2=propranolol, 3=nortriptyline,
4=amitriptyline; UHPLC instrument, X-LC with 0.1 mm i.d. connecting tube (JASCO).

Influence of frictional heating is most difficult to undergo for a core shell silica.
When using a fine particle such as 2 µm or smaller than 2 µm C18 packings underhigh pressure condition, a core shell C18 with dense end-capping showed thehighest efficiency for not only neutral compounds but also basic compounds.

Conclusion
End-capping done by both bonding of a silylation agent andconversion from silanol groups to siloxane bond leaded to
not only high efficiency for basic compounds but also
increase in retention.
Serious losses in column efficiency was caused by frictional
heating effects when very fine particles were packed into acolumn and it was operated at high back pressure. A coreshell particle showed the least losses in column efficiency,
while a totally porous hybrid particle showed large losses.
Effect of extra-column volume is important for a column
packed with very fine particles. When 0.075 mm i.d.
connecting tubes were used, SunShell C18 2 μm column
showed + 300,000 theoretical plates/meter.
In case of a low dead volume column sized 4.6 mm i.d. and150 mm length, uracil peak showed + 300,000 theoreticalplates/meter even if a particle size was 2.6 μm.
In case of 150 mm length column, efficiency was + 300,000
theoretical plates/meter even if 0.1 mm i.d. connecting
tubes were used.