YAMAZEN

ODS(C18) COLUMN
USER GUIDE
Ample information and knack on how to choose
reverse phase columns like the C18 columns.

YAMAZEN CORPORATION
 How to Use the Reverse Phase
Chromatography Column (ODS Column)?

Reverse phase chromatography is a partition

chromatography that separates a sample
based on the difference in hydrophobicity of
each compound. Normal phase
CH3 chromatography typically relies on silica gel

Si Si (CH2 )17 CH3 columns which separate a sample when the

sample is absorbed by silanol on silica gel
via hydrogen bonding. Conversely, in reverse
CH3 phase chromatography, the solid phase retains
the sample by hydrophobic interaction.
Therefore, the column selection is of critical
importance to avoid poor separations.

Sample Retention by Silica Gel Sample Retention by ODS (C18)

– Normal Phase – Reverse Phase

H CH 3


O O R Si O Si
 CH 3
H
Si Hydrogen Hydrophobic
R O
bond bond
CH3

O H H3 C Si O Si
  Bonding CH3
O O R
 strength
Si H O
CH 3
There is a hydrogen bond between Si O Si
silanol on silica gel and polar
CH 3
group on sample.
C18 alkyl chains capture the hydrophobic moieties
of sample by means of hydrophobic bond.

(1) Procedure of flash chromatography with the use of the reverse phase column 3

(2) Activation of adsorbent 4

(3) Selection of solid phase 5
(4) Sample loading method 6

(5) Method development of flash chromatography with the use of the HPLC method 7
(6) How to rinse and store the reverse phase columns
when using the same column repeatedly? 8

(7) How to choose a column (adsorbent) for reverse phase chromatography? 9

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(1) Procedure of flash chromatography with the use of
the reverse phase column
1. Activation of column
Deliver as much as 4-CV
methanol or acetonitrile.
2. Replace those solvents used for the column activation by the initial solvents
for sample run.
Deliver as much as
4-CV initial solvents.
3. Prepare proper solvents to dissolve the sample.
Dissolve sample in
initial solvents.
4. Load sample solution onto Inject Column.
Charge sample solution
onto Inject Column.
5. Start run
Bubble noise sometimes causes poor
sample detection. In that case, make the
tubing build up back pressure after UV
detector to get rid of bubble noise.
Bubble noise
C18 alkyl chains in new dry-packed ODS column
lie down and cannot capture the sample well.
(See Page 4, fig.1).
Therefore, be sure to activate the column by
delivering as much as 4-CV methanol or
acetonitrile before a sample run.
Develop the method after column activation.
Residual methanol or acetonitrile is left inside
the column that was used for column activation.
Therefore, deliver as much as 4-CV initial
solvents to completely purge the methanol or
acetonitrile.
Perform sample preparation while replacing the
residual solvents inside the column by initial
solvents. It is recommended that higher polarity
solvents be used to dissolve the sample than the
initial solvent to prevent the sample from co-eluting.
For example, the sample is run in water and
methanol, and if the sample is dissolved in
chloroform, whose polarity is lower than
water/methanol, sample will not be retained by
column sufficiently and will often lead to co-elution.
ODS columns repel water. Thus, those samples
dissolved in water will disperse poorly in a reverse
phase column. Pressing the sample into an ODS
column often leads to co-elution or poor
separation. Yamazen has a proprietary technology
to make the Reverse Phase Inject Column to allow
the reverse phase sample soak in easily.
(See Page 6, fig.2).
Mixing solvents will sometime cause bubble
generation. Air bubbles in the line will have a
detrimental effect on sample detection when
Sample size is small or the sample has low UV
absorbance. In that case, it is recommend to
degas the solvents before sample run or make
the solvent tubing build up back pressure after
UV detector so bubbles will not be generated
inside the tubing. Highly viscous samples could
build up too much pressure inside the column.
In that case, dilute the sample before loading it
onto column.
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 (2) Activation of adsorbent

C18 alkyl chains in the new column lie down and entangle each other, decreasing surface
area, which causes insufficient retention of sample and poor sample separation. Delivering
as much as 4-CV 100% methanol through a new C18 column will activate C18 alkyl chains
and assure good sample separations.

Deactivated ODS in a new column Activated ODS

C18 alkyl chains mat down on particle, C18 alkyl chains capture
which leads to insufficient retention samples efficiently by means of
and poor sample separation. the hydrophobic interaction.

Activation
Mobile phase Mobile phase

Adsorbent Adsorbent

Mobile phase comes out of pores, Pores are filled with mobile phase which
decreasing the interactive surface area. increases the number of active C18 alkyl
chains to capture the sample.

Fig.1: Difference of the result of the sample runs with or without the column activation

< Not activated > < Activated >

Uracil / Caffeine / Phenol Caffeine

Activation Uracil Phenol

This is a chromatogram without column Activation of C18 column will assure

activation. A portion of the sample is retained a good sample retention and good
by solid phase and isolated, however most of sample separation.
the sample is not retained and co-elutes.

Column: Universal Column, standard ODS, 50µm, L-size (40g)

Mobile phase: water/methanol
Flow rate: 20mL/min
Temperature: Room temperature
Sample: uracil / caffeine / phenol, 5mg/mL each (dissolved in 30% methanol / water)

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 (3) Selection of mobile phase

Water/methanol or water / acetonitrile is usually used as the mobile phase for the reverse
phase chromatography. Even with Yamazen’s end-capped ODS column, the remaining
silanol could cause peak tailing. To prevent the sample peaks from tailing, basic buffer
such as triethylamine or aqueous ammonia, or acidic buffer such as acetic acid or TFA
could be sometimes added to the mobile phase. Water / methanol is a less costly solvent
system. However higher pressure will build up inside the column when water / methanol are
used than when water / acetonitrile are used.

0.3
internal pressure (MPa)

Water / Methanol
Column's カラム圧(MPa)

0.2

Water / Acetonitrile

0.1

0
0 10 20 30 40 50 60 70 80 90 100
Ratio有機溶媒比率(%)
of organic solvent (%)

Universal Column, Standard ODS, 50µm, M-size (16g)

Flow rate: 10mL/min.
Temperature: Room temperature

Selectivity can be changed by changing mobile phase from water / acetonitrile to water
/ methanol. Those samples that are not well separated with water / acetonitrile could be
separated well if changing mobile phase to water / methanol.

Water / Acetonitrile (60/40) Water / Methanol (60/40)

Phenol
+
Benzoic acid Phenol
Change of
solvents Benzoic acid

Column: Universal Column, Standard ODS, 50µm, L-size (40g)

Flow rate: 20mL/min.
Temperature: Room temperature
Sample: phenol / Benzoic acid, 10mg/mL each (dissolved in 30% methanol / water)

Selectivity could be widely changed between methanol (protic solvent) and acetonitrile
(aprotic solvent).

-5-
 (4) Load sample solution onto Inject Column

Adsorbents used for reverse phase chromatography repel water. Thus, those samples
dissolved in waster will disperse poorly in the reverse phase adsorbents. Pressing the
sample into the reverse phase column from the top could result in rough peaks and poor
separation. Yamazen has a proprietary technology to make the Reverse Phase Inject
Column to allow the reverse phase samples soak in easily.

■Sample volume and Inject Column size

Water Water Water Water Inject Column Dissolved in Dissolved in
Size Methanol (mL) Water (mL)
2S 2 1.5
Repel S 3 2.5
M 10 7
Oil Oil Oil Oil Oil Oil
Oil Oil Oil Oil L 20 14
2L 30 21
3L 100 70
Adsorbent Adsorbent Adsorbent
Note: Samples dissolved in 100% water hardly soak in
the reverse phase adsorbent, which will decrease
the sample volume that a column can take.

Loading directly to
chromatography column Loading to Inject Column

Soak

A sample hardly soaks into adsorbent, Sample will soak into

thus sample loading takes time. adsorbent quickly.

Pressing the sample into Loading a sample by

adsorbent from top by the pipette. using an Inject Column

Fig.2: Difference in the results of the sample runs with or without Yamazen’s
proprietary Inject Column.
Rough peaks Good separation

Column: Universal Column, Standard ODS, 50µm, M-size (16g)

Mobile phase: water/methanol; Flow rate: 10mL/min.; Temperature: Room temperature

-6-
 (5) Method development of flash chromatography with
the use of the HPLC method
If the HPLC method for the reverse phase chromatography is available, the method for the
reverse phase flash chromatography can be developed by changing the mixture ratio of
methanol or acetonitrile.

<HPLC condition> ■Void Volume of HPLC Column

Column: ODS (φ4.6 x 250) HPLC Void Flow Void Time
Flow rate: 1mL/min Column Volume Rate (To)
Void time (To)： 2.9 min (See table at right.) φ2.1 x 150 0.37mL 0.2mL/min 1.85min
Method: 30% methanol in water for 5 min.
φ4.6 x 150 1.75mL 1.0mL/min 1.75min
Gradient to 60% methanol for 15 min.
60% methanol for 10 min. φ4.6 x 250 2.9mL 1.0mL/min 2.9min

HPLC condition

70
60
50
MeOH(%)

40
30
20
10
0
0 5 10 15 20 25 30
time(min)

5 min = 1.7 To 15 min = 5.2 To 10 min = 3.4 To

(5 min ÷ 2.9 min = 1.7 To)

<Flash condition> Modify the HPLC method to develop the

method for the flash chromatography.
Column: Universal Column, Standard ODS, L-size (40g)
Flow rate: 20mL/min
Void time (To)： 4 min

Flash condition

70
HPLC method
60
50
MeOH(%)

40
Method for the flash chromatography
30
20
Deduct 12% from MeOH mixture ratio applied to HPLC.
10
0
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42
time(min)

1.7 To = 7 min 5.2 To = 21 min 3.4 To = 14 min

(1.7 To × 4 min = 6.8 min)

Note: Method for the analytical HPLC is not always suitable to use to develop the method for flash chromatography.

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 (6) How to rinse and store the reverse phase columns
when using the same column repeatedly?

The reverse phase adsorbents can be used repeatedly if they are rinsed and stored properly.
The Yamazen Inject Column can function as the guard column for those samples adsorbed
by reverse phase adsorbents irreversibly and contribute to prolonged service life of the
separation column.

< How to rinse the column? >

When the sample is run in mobile phase including acid or mineral salt, be sure to
thoroughly rinse the column with mobile phase, including organic solvent without salt,
and then rinse it again with methanol or acetonitrile to store it.

< How to store the separation column? >

Do not cap the column tightly. If it is capped tightly, it might expand due to temperature
change, causing damage to it or decrease in its performance. Store the column in a place
where temperature change is minimal. When the stored column is used again, activate the
column and then replace the mobile phase with the original mobile phase before the
sample run.

< Procedure for rinsing the reverse phase column. >

The sample was run with The sample was run with the
mobile phase without salt. mobile phase including salt.

Rinse the column with the

mobile phase without salt.

Rinse the column with

methanol or acetonitrile.

Store

Replace the mobile phase in the stored

column by the mobile phase without salt.

Run a sample with mobile Run a sample with the mobile

phase without salt. phase including salt.

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 (7) How to choose a column (adsorbent) for reverse
phase chromatography?

First choice
Most popular monomeric type reverse phase adsorbent having C18
Standard ODS alkyl chains. Being end-capped, it can separate even basic
compounds without tailing.
Specifications: C18, 50µm, 120Å, carbon content 20%, end-capped

When methanol mixture ratio

is less than 5% in water Aqueous C18 column can be used with 100%
Aqueous water. This adsorbent is less hydrophobic and
works well for those compounds that cannot be
retained well by standard ODS column.
Specifications: C18, 40µm, 120Å,
carbon content 14%, end-capped

Molecular weight > 1200 This is C18 with large pore size. This adsorbent
Wide Pore works well with those compounds whose
molecular weight is larger than 1200.
Specifications: C18, 40µm, 275Å,
carbon content 18%, end-capped
For those samples whose
hydrophobicity is extremely
high and are captured too
strongly by C18 alkyl chains. C8 and C4 have shorter hydrophobic chains
Octyl (C8) than C18 and will work well for those samples
that have more hydrophobic moieties to interact
Butyl (C4) with the column.
Specifications: C8 or C4, 40µm, 60Å or 275Å,
end-capped
When it is necessary to make
mobile phase for strong
acidity or strong alkalinity,
or utilizing the capability of
recognizing planar molecule. Polymeric type of C18. As compared to
Polymeric monomeric type of C18, polymeric type of C18
can better withstand strong acid and strong
basic. Polymeric type of C18 has a better
capability of recognizing planar molecule,
and allows a change in eluting pattern for
planar molecule and non-planar molecule.
Specifications: C18, 50µm, 120Å,
carbon content 20%, no endcap.
When changing selectivity These adsorbents are non-end-capped C18.
Polar Plus Remaining silanol will contribute to sample
separation.
Light Load Specifications: C18, 40µm, 60Å,
carbon content 16% or 20%,
no endcap.
When enhancing the selectivity
for aromatic compounds Phenyl adsorbent separates compounds based
Phenyl (C6) on hydrophobic interaction and tt-tt interaction,
enhancing selectivity for aromatic compounds.
Using methanol in mobile phase will make it
easy to utilize the tt-tt interaction.
Specifications: C6, 40µm, 60Å, end-capped

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YAMAZEN CORPORATION
HEAD OFFICE RECRUIT SHINOSAKA BLDG. 3F, 5-14-22 NISHINAKAJIMA,
YODOGAWA-KU, OSAKA 532-0011, JAPAN
TEL: +81-6-6304-5839 FAX: +81-6-6304-3681
R&D SANWA BLDG. 101, 4-6-10 NISHINAKAJIMA,
YODOGAWA-KU, OSAKA 532-0011, JAPAN
TEL: +81-6-6304-7284 FAX: +81-6-6304-7283
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TEL: +81-3-5256-6481 FAX: +81-3-5256-6480
E-MAIL info@yamazenc.co.jp
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November 2014