HPLC Column

Technical Guide

(1) Introduction P1

(2) Specifications P1～4

(3) Shipping Solvent P5～8

(4) Mobile Phase P 9 ～ 11

(5) Sample Diluent/Injection Solvent P 12

(6) Column Cleaning and Storage P 13 ～ 17

(7) Tips to Increase HPLC Column Lifetime P 18 ～ 21

(8) Benefits of Scaling Down Column Internal Diameter Size P 22 ～ 26

(9) Tips on Maximizing Efficiency on UHPLC Columns P 27 ～ 28

(10) Benefits of Metal-Free PEEK Columns P 29 ～ 30

(11) Tips on Maximizing Purification Efficiency on Preparative Columns P 31 ～ 33

22-1 Nishishinjuku, 6-Chome

Shinjuku-ku, Tokyo 163-1130, Japan
TEL: +81-35323-6620
FAX: +81-35323-6621
Web: [Link]
Email: world@[Link]
(1) Introduction

Thank you for choosing GL Sciences’ HPLC columns. GL Sciences’ HPLC columns are subjected
to a rigorous array of QC test in the ISO 9001 compliant facility, with special emphasis on
reagent purity, raw material traceability and consistency in raw materials, and final products.
To maintain and maximize peak performance of GL Sciences’ HPLC columns, and ensure long
life and stability, please read the following before use.

(2) Specifications

(2-1) Recommended Operating Pressure (Maximum Operating Pressure)

GL Sciences’ HPLC columns can tolerate up to pressures shown below. Although the columns
are packed by high pressure slurry method, it is recommended to keep the operating pressure
under the pressure shown below to maintain peak performance and ensure long column life
and stability.

●Recommended Operating Pressure for Analytical Columns

Recommended
Analytical C olumns Particle Size Operating Pressure
(MPa)

Inertsil series, InertSustain series,

1.9 μm, 2 μm 80
InertSustainSwift series

Inertsil series, InertSustain series,

3 μm HP 50
InertSustainSwift series

Inertsil series, InertSustain series,

3～10 μm 20
InertSustainSwift series, Titansphere

InertC ore C 18 2.4 μm 100

InertSphere Sugar-1 5 μm 15

C apillary EX columns 3 μm, 5 μm 20

C apillary EX Nano columns 3 μm, 5 μm 15

●Recommended Operating Pressure for Guard Columns

Recommended
Guard C olumns Operating Pressure
(MPa)

Guard C olumn for UHPLC 80

C artridge Guard C olumn E

C artridge Guard C olumn Ei
20
GL-C art, Pre-clean ORG
PREP Guard C artridge

C onventional Guard C olumn

C onventional Mini Guard C olumn
20
C apillary Micro Guard
Preparative Guard C olumn
1
(2-2) Temperature and pH Range

The recommended operating pH range and temperatures are shown below. To maximize column
lifetime, set the pH of the mobile phase within the range shown below. Additionally, use mixed
mobile phase such as organic solvent and buffer rather than 100 % buffer. When operating at
high pH, lower operating temperatures are recommended for longer column lifetime. Working at
high temperatures may also result in shorter column lifetimes. Please note that the column
lifetime will vary depending upon the operating temperature, the type, and concentration of
buffer used.
pH Range

Maximum
C olumns Operating 20 ～ 40 ℃ 40 ～ 50 ℃ 50 ～ 60 ℃
Temperature

Inertsil series, Titansphere 2 - 7.5

InertSustain C 18, InertSustain AQ-C 18

1 - 10 (*) 1-7
InertSustainBio C 18, InertSustainSwift C 18
InertSustain C 8, InertSustain Phenylhexyl,
1 - 10 (*) 1 - 9 (*) 2-7
InertSustainSwift C 8
60 ℃
InertSustain Amide 2 - 8.5 (*) 2-7

InertSustain Phenyl, InertSustain NH2 2 - 7.5

InertC ore C 18 2 - 7.5

InertSphere Sugar-1 80 ℃ 2 - 14 (5 ～ 80 ℃)

* For method development at low pH (pH 1 ～ 2), the usage of either trifluoroacetic acid, formic acid,
acetic acid or phosphate buffer is recommended. At high pH (pH 10), the buffer concentration should
be adjusted at 5 mM using buffers such as trimethylamine. In case an organic solvent is not present
in the buffer mobile phase, set the pH in the range within 2 ～ 8.
Lower operating temperatures are recommended for longer column lifetime at pH 1 ～ 2 or pH 9 ～
10. Additionally, it is recommended to have an organic solvent (e.g., methanol) present in your
mobile phase.

2
(2-3) Column End-fittings

The specification of GL Sciences’ end-fitting styles are summarized below. The chromatographic
separation and result can be negatively impacted if the style of the column end-fittings does not
match the existing tubing ferrule settings.

●End-fitting Styles for Analytical Columns

Analytical C olumns Particle Size End-fitting Style

InertSustain AQ-C 18 1.9 μm UP Type

InertSustainSwift C 18, InertSustainSwift C 8 1.9 μm 、3 μm HP UP Type

InertSustain C 18, InertSustain C 8, InertSustain Phenyl

2 μm W Type
Inertsil series

InertSustain AQ-C 18, InertSustainC 18, InertSustain C 8,

InertSustain Phenylhexyl, InertSustain Phenyl,
3 μm HP、3 ～ 10 μm W Type
InertSustain Amide, Inertsil series, Unisil Q series,
Titansphere

UHPLC PEEK, PEEK columns All Particle Sizes UP Type

InertC ore C 18 2.4 μm UP Type

InertSphere Sugar-1 5 μm W Type

C apillary EX columns 3 μm 、 5 μm UP Type

C apillary EX Nano columns 3 μm 、 5 μm UP Type

* Each end-fitting style differs in the required length of the tubing protruding from the ferrule.
The UP type requires a 2.4 mm ferrule depth.
The W type requires a 3.3 mm ferrule depth.
For more details, please see page 5.

3
●End-fitting Styles for Guard Columns

Guard C olumns End-fitting Styles

C artridge Guard C olumn E

C artridge Guard C olumn Ei
W Type
GL-C art, Pre-clean ORG
PREP Guard C artridge

Guard C olumn for UHPLC UP Type

C onventional Guard C olumn

C onventional Mini Guard C olumn
W Type
C apillary Micro Guard
Preparative Guard C olumn

●Various End-fitting Styles

Various column manufacturers have employed different types of chromatographic column
connectors. The following explains the differences in each type.

● W Type ● UP Type (Parker style) ● OS Type

10-32UNF 10-32UNF 10-32UNF

3.3 m m 2.4 m m 2.4 m m

● T Type ● ON Type ● HA Type

10-32UNF 10-32UNF 10-32UNF

2.7 m m 2.7 m m
4.4 m m

4
 (3) Shipping Solvent

(3-1) Various Shipping Solvents

GL Sciences’ HPLC columns are shipped in the solvents shown below. Please be reminded that
normal-phase columns are shipped in non-aqueous solvents.

C olumns Particle Size, Dimensions Shipping Solvent

・InertSustain C 18, C 8, AQ-C 18,
Phenylhexyl, Phenyl、Amide
・InertSustainSwift C 18, C 8
・Inertsil ODS-3V, ODS-4V, Amide, HILIC ,
ODS-HL、ODS-P、ODS-EP、ODS-80A、 All Particle Sizes
Reversed-Phase
C 8-4, C 8-3, C 8, C 4, Ph-3, Ph, & Acetonitrile/Water
C olumns
WP300 C 18, WP300 C 8, WP300 C 4, All Dimensions
ODS, C 30, Peptides, Acrolein, Sulfa
・InertC ore C 18
・MonoTower C 18, MonoC lad C 18-HS

5 µm 250 x 4.6 mm I.D.

Reversed-Phase Methanol/Water
・Inertsil ODS-4, ODS-3, ODS-SP, ODS-2 5 µm 150 x 4.6 mm I.D.
C olumns

Other Particle Sizes, Dimensions Acetonitrile/Water

・InertSustain NH2
All Particle Sizes
Normal-Phase ・Inertsil NH2、Diol、C N-3、SIL-100A、
& n-Hexane/Ethanol
C olumns SIL-150A、WP300 SIL、WP300 Diol,
All Dimensions
・Titansphere TiO
All Particle Sizes
Ion-Exchange
・Inertsil AX、 C X & 100 % Methanol
C olumns
All Dimensions
All Particle Sizes 100 mM
Polymer C olumns ・InertSphere Sugar-1 & Sodium Hydroxide
All Dimensions Aqueous Solution

(3-2) Column Equilibration Prior to Use

It is important to first understand the mobile phase compatibility before changing to a different
mobile phase system. This understanding will help to avoid precipitating mobile phase buffers
on your column as well as in your system. Before injecting the sample, thoroughly equilibrate
the column with the mobile phase to be used to ensure stable chromatographic separation. For
more details, please refer to the following.

●Column Equilibration Procedure for Reversed-Phase Columns (C18)

If the mobile phase does not contain buffers or additives, equilibrate the column and system
with the mobile phase to be used for at least 30 minutes. The column may be considered
equilibrated once a steady column back pressure and baseline are observed.
If the mobile phase contain buffers or additives, equilibrate the column and system with a
water/organic solvent mixture, using the same content as in the buffered mobile phase. For
example, equilibrate the column and system with 20 % acetonitrile in water for at least 30
minutes. Then, introduce and equilibrate with 20 % acetonitrile / 80 % buffered mobile
phase for at least 30 minutes. The column may be considered equilibrated once a steady
column back pressure and baseline are observed.

●Column Equilibration Procedure for Normal-Phase Columns

If the mobile phase does not contain buffers or additives, equilibrate the column and system
with the mobile phase to be used for at least 30 minutes. The column may be considered
equilibrated once a constant column back pressure and baseline are observed.
5
 If the mobile phase contain buffers or additives, equilibrate the column and system with a
mixture of solvents, using the same content as in the buffered mobile phase. For example, if
the mobile phase is n-hexane / ethanol / acetic acid (900 /100 / 1), equilibrate the column
and system with n-hexane / ethanol (900 / 100) for at least 30 minutes. Then, introduce and
equilibrate with the mobile phase to be used for at least 30 minutes. The column may be
considered equilibrated once a steady column back pressure and baseline are observed.
When using normal-phase column for reversed-phase methods, the column must be properly
equilibrated before use. For more details, please refer to section (3-3).

●Column Equilibration Procedure for HILIC Columns

(InertSustain Amide, Inertsil Amide, Inertsil HILIC)
If the mobile phase does not contain buffers or additives, equilibrate the column and system
with the mobile phase to be used for at least 120 minutes. The column may be considered
equilibrated once a constant column back pressure and baseline are observed.
If the mobile phase contain buffers or additives, equilibrate the column and system with a
water/organic solvent mixture, using the same content as in the buffered mobile phase. For
example, if the mobile phase is 90 % acetonitrile / 10 % ammonium acetate aqueous
solution, equilibrate the column and system with 90 % acetonitrile in water for at least 30
minutes. Then, introduce and equilibrate with the mobile phase for at least 120 minutes. The
column may be considered equilibrated once a steady column back pressure and baseline are
observed.

●PEEK and UHPLC PEEK Columns

Avoid the use of tetrahydrofuran and chloroform as it can weaken the PEEK hardware and
cause it to become brittle. The column equilibration procedure shall be the same as a
stainless steel hardware column prior to the analysis.

6
(3-3) Column Equilibration Procedure for Normal-Phase Columns to be used for
Reversed-Phase Methods
GL Sciences’ normal-phase columns can be used for both normal-phase and reversed-phase
separations. As shown at section (3-1), these columns are originally shipped in non-aqueous
solvents and is ready to use for normal-phase conditions. If you intend to use the column for
reversed-phase separations, you will require to condition the column with the following
procedure:

【 For Inertsil NH2 (5 μm, 150 x 4.6 mm I.D.) 】

a) Flush the column and system with isopropyl alcohol at 0.5 mL/min for at least 60 minutes.
b) If the mobile phase contain buffers or additives, equilibrate the column and system with a
water/organic solvent mixture, using the same content as in the buffered mobile phase. For
example, equilibrate the column and system with 90 % acetonitrile in water at 0.5 mL/min
for at least 60 minutes.
c) Then, introduce and equilibrate with 90 % acetonitrile / 10 % buffered mobile phase for at
0.5 mL/min for at least 60 minutes.
d) The column may be considered equilibrated once a steady column back pressure and
baseline are observed.
e) Finally, inject the sample several times and make sure the retention times are not shifting.

● Difference in Column Stability depending on the Column Equilibration Procedure

As illustrated in the following page, the shift in retention times are not occurring in Figure B,
which the column was properly equilibrated. Columns that were improperly equilibrated will
most likely show results illustrated in Figure A.

7
HPLC Conditions
Column : Inertsil NH2
Sample :
(5 μm, 150 x 4.6 mm I.D.)
1. Fructose
Eluent : A) CH3CN
2. Glucose
B) H2O
3. Sucrose
A/B = 75/25, v/v
4. Maltose
Flow Rate : 1.0 mL/min
5. Lactose
Col. Temp. : 40 ℃
Detection : RI

Figure A. Figure B.

The column was equilibrated only with a The column was properly equilibrated as per
water/organic solvent mixture, using the the procedure described in page 8, section
same content as in the mobile phase. In (3-3).
other words, the column was not initially
flushed with isopropyl alcohol.

1 1
2 3 2 3
4 5 4 5
Equilibration Time Equilibration Time
120 min 120 min

90 min 90 min

60 min 60 min

30 min 0 2 4 6 8 10 12 30 min 0 2 4 6 8 10 12
Time Time
(min) （min
Stable
Unstable even after 2 hours

8
(4) Mobile Phase

(4-1) Various Grades of Solvents

To maintain maximum column performance, use high quality HPLC or MS-grade solvents.
Solvents containing suspended particulate materials will clog the outside surface of the inlet frit
of the column. This will result in higher operating back pressure. Additionally, the amount of
contaminants/impurities or stabilizing agents (additives) contained in the solvents depends on
the grades of solvents. These contaminants and stabilizing agents do have UV (ultraviolet)
absorbance resulting in interfering chromatographic results. Select the appropriate grade of
solvent depending on your method and application.

The UV absorbance curve is shown below between various grades of solvents.

●Methanol

Blue HPLC-Grade
Green Special Grade
Red 1st Grade
A significant difference in UV
absorbance was not observed.

●Acetonitrile

Blue HPLC-Grade
Green Special Grade
Red 1st Grade
Both special and 1st grade solvents
showed absorbance at lower
wavelengths.

●THF (Tetrahydrofuran)

Blue HPLC-Grade When using THF, make sure using an HPLC

Green Special Grade grade. Other grades of THF contain
Red 1st Grade
antioxidants that can be detected by UV
detectors and therefore affect the UV
background. Additionally, the use of old THF
may also result in a high UV background due
to the formation of peroxides.

9
 (4-2) Degassing Mobile Phase Solvents
When solvents are in contact with the atmosphere, air gradually dissolves into the solvent. The
amount of dissolved air in solvents depends on the composition of mobile phase, temperature,
and pressure. When the aqueous and organic solvents are mixed, they each contribute to the
total dissolved air content of the mixture. Whether we are making isocratic mixtures or
gradients, the amount of gas in solution is in proportion to the respective solvent volumes. The
problem with this situation is that the solubility of air in the mixture is less than that of
individual components. When such a situation exists, the solution is supersaturated with air,
generating an unstable condition in which air will bubble out from the solution resulting in
causing the following problems.

●Problems Possibly Caused by Insufficient Degassing of Solvents

Places where air

bubbles may Problematic Symptoms Estimated Chromatographic Results
have formed

Unstable flow of solvent. Shift in retention times.

Pump
Fluctuation of flow of solvent. Change in peak areas.

Distorted peak shapes.

Column Decrease in column efficiency.
Less column efficiency.

Baseline drift or baseline noise.

Detector Interference on detection of analyte.
Decrease in sensitivity of detector.

●Degassing Methods
The following describes the degassing methods and their features.

Methods Operation Procedures Features

Attach an aspirator to the mobile phase bottle and

Can be done at a low cost, but the
Vacuum degas by vacuuming for at least 15 minutes while
solvent content can be changed.
agitating the mobile phase to remove air bubbles.

Place the mobile phase bottle into an ultrasonic Safe and easy, but air bubbles cannot be
Sonication
cleaner and sonicate for at least 10 minutes. removed completely.

Vacuum Place the mobile phase bottle into an ultrasonic The solvent can be degassed in a short
+ cleaner and attach an aspirator. Vacuum for at least time, however, the solvent content can
Sonication 2 minutes while sonicating the solvent. change very easily.

Very effective and can reduce the

dissolved air in common solvents to
A stream of helium bubbles will sweep dissolved air
Helium Sparge levels below the saturation level of
out of solvents.
mixtures. However, the cost of helium
gas is high.

A membrane degasser uses a tube made of semi-

permeable membrane passing through a vacuum Easy-to-use and the solvent content does
Degasser
chamber. Gasses diffuse through the membrane and not change so much.
are removed. Solvents are retained in the tube.

10
● Cautions on Degassing
Low temperature promotes gas solubility. Therefore, solvents stored at a cold place may
contain a large volume of air. Additionally, air may be present in a mixture of
acetonitrile/water as the temperature decreases after mixing these two solvents. Solvents
that cool overnight should be stabilized to room temperature and fully degassed prior to
introducing work into the system.

11
 (5) Sample Diluent

(5-1) The Effect of Difference in Elution Strength between Sample Diluent and Mobile
Phase
Ideally, the sample diluent should have a composition as close as possible to that of the mobile
phase used for the separation i.e. the diluent should match the initial mobile phase conditions.
As illustrated below, injecting a solvent stronger than the mobile phase can cause peak shape
problems, such as peak splitting.

Sample Diluent : 100 % Acetonitrile Sample Diluent : Mobile Phase

1
2

1 3
3 4 4

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Time (min) Time (min)

HPLC Conditions Sample

Column : InertSustain AQ-C18 (5 μm, 150 x 4.6 mm I.D.) 1. 5-Hydroxymethyl-2-furaldehyde
Eluent : A) CH3CN 2. 2-Furfural
B) H2O 3. 2-Acetylfuran.
A/B = 10/90 , v/v 4. 5-Methyl-2-furfural
Col. Temp. : 40 ℃
Detection : UV 280 nm
Injection Volume : 10 μL

(5-2) The Effect of Difference in pH between Sample Diluent and Mobile Phase
Peak splitting or fronting of peaks may be observed when the pH of the sample diluent and that
of mobile phase are too different. Such problem can be overcome by diluting the sample
solution with the mobile phase or decreasing the injection volume.

12
(6) Column Cleaning and Storage

Residual ion-pairing reagents, acids, or buffer salts in the column will promote and encourage
hydrolysis of the bonded phase resulting in shorter column lifetimes. Additionally, storage in
highly aqueous mobile phases for a long period can lead to splitting peaks or less column
efficiency as the sample matrices such as lipids can accumulate in the column. To prolong
column lifetimes, clean the column every single time after the analysis or whenever possible.

(6-1) Cleaning Reversed-Phase Columns

● When the mobile phase does not contain any buffered mobile phases or ion-pairing reagents
Use high concentration organic solvent to remove the highly lipophilic contaminants. Increase
the content of organic solvent up to 100 %. Then, flush the column with 5 column volumes.
When observing excessive back pressure, reduce and adjust the flow rate.

Example Column Dimension 4.6 mm I.D. x 250 mm

Method Flow Rate 1 mL/min
Method Mobile Phase acetonitrile/water = 65/35

Step 1 : Clean the column with 100 % acetonitrile at 1 mL/min for at least 30 minutes.

● When the mobile phase contain buffered mobile phases

Clean the column with a water/organic solvent mixture, using the same content as in the
buffered mobile phase. For example, clean the column with 20 % acetonitrile in water for at
least 30 minutes. Then, clean it with 100 % acetonitrile.

Example Column Dimension 4.6 mm I.D. x 250 mm

Method Flow Rate 1 mL/min
Method Mobile Phase 10 mM KH2PO4/acetonitrile ＝ 80/20

Step 1 : Clean the column with 20 % acetonitrile in water at 1 mL/min for at least 30 minutes.
Step 2 : Clean the column with 100 % acetonitrile at 1 mL/min for at least 30 minutes.

* When using the column again for the analysis, follow the procedures below to avoid
precipitating mobile phase buffers on the column.

Step 1 : Equilibrate the column with 20 % acetonitrile in water at 1 mL/min for at least 30
minutes.
Step 2 : Equilibrate the column with the buffered mobile phase to be used at 1 mL/min for at
least 30 minutes.
Step 3 : The column may be considered fully equilibrated once a constant back pressure and
stable baseline are observed.

13
● When the mobile phase contain ion-pairing reagents
Depending on the ion-pairing reagent type, precipitation may occur when cleaning the column with
100 % water and extreme care is required. Clean the column with a water/organic solvent mixture,
using the same content as in the mobile phase containing an ion-pairing reagent. For example,
clean the column with 10 % acetonitrile in water for at least 30 minutes. Then, clean it with
water/acetonitrile = 50/50 for at least 30 minutes.
The content of organic solvent should be increased further when using ion-pairing reagents
containing long alkyl chains to effectively remove out from the column.

Example Column Dimension 4.6 mm I.D. x 250 mm

Method Flow Rate 1 mL/min
Method Mobile Phase 10 mM KH2PO4 + 2 mM *IPCC-09 (pH:2.5)/acetonitrile = 90/10

Step 1 : Clean the column with 10 % acetonitrile in water at 1 mL/min for at least 30 minutes.
Step 2 : Clean the column with acetonitrile/water = 50/50 for at least 30 minutes.

* IPCC-09： Sodium 1-nonanesulfonate

* Please be aware that removal of 100 % of the ion-pairing reagent may not be possible. Due to the
fact that ion-pairing reagents can alter column selectivity, it is strongly recommended to dedicate
columns to ion-pairing methods to avoid problems with reproducibility.

14
(6-2) Cleaning HILIC Columns
Under HILIC mode, polar analytes are retained with high organic mobile phases. The following
describes the elution strength of solvents used in HILIC mode.

Tetrahydrofuran < Acetonitrile < 2-Propanol < Ethanol < Methanol < Water
Weak Solvents Strong Solvents

● Cleaning InertSustain Amide Columns

To avoid precipitating mobile phases buffers within the column, clean the column with a
water/organic solvent mixture, using the same content as in the buffered mobile phase. Clean the
column with acetonitrile / water = 50 / 50 to remove highly polar contaminants.
If the column still shows shift in retention time or distorted peak shapes, clean the column with
100 % water for at least 30 minutes. After cleaning the column, make sure to thoroughly
equilibrate the column with the mobile phase to be used in the analysis prior to use. Store the
InertSustain Amide column in 100 % acetonitrile.

Example Column Dimension 4.6 mm I.D. x 250 mm

Method Flow Rate 1 mL / min
Method Mobile Phase 5 mM CH3COONH4 / acetonitrile = 10 / 90

Step 1 : Clean the column with acetonitrile / water = 90 / 10 at 1 mL / min for at least 30 minutes.
Step 2 : Clean the column with acetonitrile / water = 50 / 50 at 1 mL / min for at least 30 minutes.

15
●Cleaning Inertsil HILIC Columns
To avoid precipitating mobile phases buffers within the column, clean the column with a
water / organic solvent mixture, using the same content as in the buffered mobile phase. Clean
the column with 100 % water to remove highly polar contaminants.

Example Column Dimension 4.6 mm I.D. x 250 mm

Method Flow Rate 1 mL / min
Method Mobile Phase 5 mM CH3COONH4 / acetonitrile = 10 / 90

Step 1 : Clean the column with acetonitrile / water = 90 / 10 at 1 mL / min for at least 30 minutes.
Step 2 : Clean the column with 100 % water in water at 1 mL / min for at least 30 minutes.

●Cleaning InertSustain NH2 Columns

To avoid precipitating mobile phases buffers within the column, clean the column with a
water / organic solvent mixture, using the same content as in the buffered mobile phase. Clean
the column with acetonitrile / water = 50 / 50 to remove highly polar contaminants.
If the column still shows shift in retention time or distorted peak shapes, clean the column
with 50 mM ammonium formate (or ammonium acetate) aqueous solution / acetonitrile =
50 / 50 for at least 30 minutes. After cleaning the column, make sure to thoroughly equilibrate
the column with the mobile phase to be used in the analysis prior to use. Store the
InertSustain NH2 column in 100 % acetonitrile.

Example Column Dimension 4.6 mm I.D. x 250 mm

Method Flow Rate 1 mL / min
Method Mobile Phase 5 mM CH3COONH4 / acetonitrile = 10 / 90

Step 1 : Clean the column with acetonitrile / water = 90 / 10 at 1 mL / min for at least 30 minutes.
Step 2 : Clean the column with acetonitrile / water = 50 / 50 at 1 mL / min for at least 30 minutes.

16
(6-3) Cleaning Normal-Phase Columns
Normal-phase separations depend upon polar adsorptive interactions, which the bonded
phase is polar and the mobile phase is non-polar. Polar analytes will be more strongly
retained than non-polar analytes in normal-phase chromatography. Clean the column with
polar solvents to remove highly polar contaminants.
The following describes the elution strength of solvents used in normal-phase mode.

Hexane < Chloroform < Tetrahydrofuran < 2-Propanol < Ethanol

Weak Solvents Strong Solvents

●Cleaning Inertsil SIL-100A, Inertsil SIL-150A, Inertsil WP300 SIL, Inertsil NH2, Inertsil CN-3,
Inertsil Diol, InertSustain NH2 Columns
Clean the column with ethanol or 2-propanol. Because alcohol solvents are quite viscous,
adjust the flow rate to avoid excessive column back pressure.

Example Column Dimension 4.6 mm I.D. x 250 mm

Method Flow Rate 1 mL / min
Method Mobile Phase n-hexane / 2-propanaol / acetic acid = 90 / 10 / 0.1

Step 1 : Clean the column with 100 % 2-propanol at 0.2 mL/min for at least 60 minutes.

(6-4) Column Storage

Residual ion-pairing reagents, acids or salts in the column will promote and encourage
hydrolysis of the bonded phase resulting in shorter column lifetimes. Additionally, storage in
highly aqueous mobile phases for a long period can lead to splitting peaks or less column
efficiency as the sample matrices such as lipids can accumulate in the column. To prolong
column lifetimes, clean the column every single time after the analysis or whenever possible.
After cleaning the column properly (refer to section 6-1 to 6-3), store the column by referring
to the following table.

Storage longer than a

Bonded Phase Storage from 1～10 Days
few weeks
ODS, C8, C4, Ph Mobile Phase
100 % Acetonitrile
HILIC, Amide (without salts, additives)

Mobile Phase
CN, NH2, Diol, SIL 100 % n-Hexane
(without salts, additives)

Mobile Phase
AX, CX 100 % Acetonitrile
(without salts, additives)

＊ Columns should be stored in a cool and dark place.

＊ Columns stored for a long period of time should be cleaned prior to use.

17
(7) Tips to Increase HPLC Column Lifetime

To increase column lifetime, follow these important guidelines.

(7-1) Column Handling

●Do not drop or bump columns, to avoid a deterioration of the column performance. Make sure
to disconnect the column from the system after confirming the display of the pressure gauge
showing zero “0” value. Avoid rapid pressure fluctuation to extend column lifetime.

●Use a pump equipped with an inline filter to prevent particulates from worn pump seals or
contaminants from mobile phases entering the column.

●To maximize column lifetime, use the column within the operating conditions described at
section (2). Please note that working in combinations of extreme pH, temperature and
pressure will result in shorter column lifetime.

●Residual ion-pairing reagents, acids or salts in the column will promote and encourage
hydrolysis of the bonded phase resulting in shorter column lifetimes. To prolong column
lifetimes, clean the column every single time after the analysis or whenever possible. For
more details, please read section (6).

●Only use ultrapure water in the mobile phase. Routinely maintain the water purification
system to ensure it is functioning properly. The usage of contaminated water can cause noisy
or drifting baselines and detecting ghost peaks, which are frequently observed in gradient
methods. Additionally, use freshly prepared purified water and avoid storing ultrapure water
(or aqueous solvents). Ultrapure water will absorb contaminants from the laboratory
atmosphere and from containers.

● Comparison of Residual Contaminants in Water between various Water Purification Process

0 1 20 0 1 20 0 1 20 0 1 20
0 min 0 min 0 min 0 min

Ultrapure Water Purified Water Distilled Water Ion-Exchanged Water

18
 (7-2) Guard Columns
Guard columns are connected in between the sample injector and analytical column to protect
analytical column against contamination by sample particulates and strongly retained
compounds. GL Sciences offer two different types of guard columns, a cartridge format
requiring an exclusive holder and packed with the same material as in the analytical column.
The packed guard column type is packed with the same material as in the analytical column too,
but to a stainless steel hardware format.

●Selecting the Appropriate Column Protection System

It is best to choose a guard column that contains the same packing material as your
analytical column. The monolithic silica type guard cartridge, SILFILTER STD C18 is
compatible and can be used with other C18 analytical columns.

●Selecting the Appropriate Guard Column Size

Particle Size : Select a guard column that contains the same packing
material and particle size as your analytical column.
Internal Diameter : Select a guard column with the same I.D. size or similar as
your analytical column.

Format Product Analytical Guard Column Guard Column

Column I.D. I.D. Length

Cartridge Guard Colum E 1.0 ～ 2.1 1.0 ～ 1.5

(First Choice Guard Column)
10 mm
mm mm

Cartridge Guard Column Ei 2.1 ～ 4.6 2.1 ～ 4.0

(Metal-Free Guard Column) 10、20 mm
mm mm

Cartridge Type
An Exclusive Holder is Guard Column for UHPLC 1.0 ～ 3.0 1.0 ～ 3.0
10 mm
Required (Can Tolerate Pressures up to 80 MPa) mm mm

SILFILTER STD C18

TM
3.0 ～ 4.6
(Compatible with Other C18 Columns) 3.0 mm 10 mm
mm

GL-Cart
(Available only in one size, but Economical)
4.0、4.6 mm 4.6 mm 5 mm

Packed Guard Column 1.0 ～ 4.6 1.0 ～ 4.6

(Packed to a Stainless Steel Hardware)
33、50 mm
mm mm

Packed Guard Column

Packed Mini Guard Column 4.0、4.6 mm 4.0 mm 10 mm
Type
(Short Size)
Holder Not Required

6.0 ～ 100 6.0 ～ 100 50 mm

Preparative Guard Column 75 mm (For 50 mmI.D.)
mm mm
100 mm (For 100 mmI.D.)

●Guard Column Installation

Column connectors and tubings are required separately to connect guard columns to
analytical columns. Make sure to select the appropriate connectors depending on the type of
end-fittings used on the analytical column, otherwise, chromatographic performance will be
negatively impacted due to extra dead volume.
19
 ●Type of End-fittings
End-fitting styles differ among various manufacturers and have different seating depths.
Always make sure to select the appropriate end-fittings, otherwise, chromatographic
performance will be negatively impacted due to extra dead volume. For more details on the
end-fitting styles, please see section (2-3).

●Proper Column Connection ●Improper Column Connection

Male nut side W Type Column side W Type Male nut side UP Type
Column side W Type

No Void Void

●Influence of Extra Dead Volume on Chromatographic Performance

140
120 2
100 Column Injector 1
80
mAU

60
40
20
0
0 2 4 6 8 1
0
Time (min)
140
120 Column Injector
100 2
80
1
mAU

60
40
20
0
0 2 4 6 8 1
0
Time (min)

(7-3) Sample Preparation

●Sample Filtration
Particulates often contribute to column contamination. To maximize column lifetime, filtrate
sample through a disposable syringe filter to remove particulates.

20
●Sample Preparation for Biological Samples
Proteins are present in biological samples which can interfere the quantitation of the target
analytes. Additionally, the protein matrices can reduce the column lifetime, especially under
reversed-phase HPLC. In some cases, some target analytes can bond with protein matrices
leading to a negative impact on the accuracy, precision, and robustness of the method.

●Major Deproteinization Methods

《Protein Precipitation》
Add organic solvent to the biological sample to denature it. Centrifuge the precipitated
sample solution. Take the supernatant portion of the sample and then filtrate.

《Ultrafiltration》
The primary basis for separation is molecular size. Proteins, which are larger than the
membrane pores will be retained at the surface of the membrane.

《Protein Hydrolysis》
Protein hydrolysis is the breakdown of protein into smaller peptides and free amino acids
using enzymes.

21
(8) Benefits of Scaling Down Column Internal Diameter Size

By scaling down to a narrower internal diameter column, we can significantly reduce the flow
rate and reduce costs by reducing solvent consumption. Depending on the detector type,
improvement in sensitivity can be obtained.
However, caution needs to be taken when scaling down the column. The following adjustments
are required to maximize the column performance.

(8-1) Adjust the Flow Rate (Linear Velocity)

To maintain equivalent separation and
retention times when transferring a method it is
important to keep the linear velocity constant ID 4.6 mm
1 mL/min
between the original and new method.
The linear velocity is related to the flow rate and
Both of the Linear Velocity are 1 mm/sec
internal diameter of the column.

Flow Rate (mm3/s)

Linear Velocity (mm/s) = ID 3.0 mm
Column Cross-Sectional
Area (mm2) 0.4 mL/min
Inlet Outlet

2 2 HPLC Conditions
System ：GL7700 HPLC
80

Column : InertSustainSwift C18

1 4 4
40 60

(3 μm, 150 mm)

mAU

mAU

1
100

Eluent : A) CH3CN
B) H2O
3 3 A/B = 65/35, v/v
Column Temp. : 40 ℃
20

Detection : UV 254 nm
Injection Vol. : 2.0 μL
0

0 2 4 6 8 0 2 4 6 8
Time (min) 1. Acetophenone
Time (min) 2. Benzene
3. Toluene
4. Naphthalene
ID 4.6 mm ID 3.0 mm
Flow Rate 1.0 mL/min Flow Rate 0.4 mL/min

60 % Less Solvent Consumption

Ratio of Column
1 Cross-Sectional 0.4
Area

22
(8-2) Gradient Delay
A low pressure gradient mixing system (quaternary pump) has one pump, which is used to
deliver the mobile phase to the system. Caution needs to be taken when scaling down the
column internal diameter size.

Low Pressure The point where mixing of the solvents begins is right before the pump
Gradient Mixing unit. Once the solvents are delivered to the pump, the plunger draws the
System solvent into the pump head(s) and creates a turbulent environment where
the mobile phase mixes together.
Mixing
point of
solvents Because the mobile phase is not under pressure at the mixing point of
solvents, quaternary pumps are typically considered low pressure gradient
mixing system.

A B C D

High Pressure In a high pressure gradient mixing system, we can prepare one solvent
Gradient Mixing per pump, with each pump providing flow for a specific solvent. The
System solvents are then combined in a mixing chamber that is located after the
Mixing pumps. This creates a high pressure proportioning environment because
point of the solvents are already under pressure before they reach the mixing
solvents point where the mixing occurs.

A B C

●Variation in Gradient Delay Volume between Low/High Pressure Gradient Systems

The biggest impact these two mixing modes have on chromatography is on the gradient delay
volume, specifically on the mixer and tubing volume. Gradient delay volume is measured
from where the mobile phase begins mixing to where it reaches the column. Generally, low
pressure gradient system shows larger gradient delay volume as the mixing of the mobile
phase begins is right before the pump unit. As shown below, the gradient delay can be
observed more on the low pressure gradient system when using narrower ID size at low flow
rates.

Column ID 4.6 mm Column ID 3.0 mm HPLC Conditions

(Flow Rate 0.42 mL/min) Column : InertSustainSwift C18
(Flow Rate 1.0 mL/min)
Standard 3 μm, 150 x 4.6 mm I.D.
High High 1 Semi-micro 3 μm, 150 x 3.0 mm I.D.
1 2
Pressure 2 Pressure
34 Eluent : A) CH3CN
Gradient 34 Gradient 5 B) H2O
5 A/B = 50/50 - 6min – 100/0, v/v

Column Temp. : 40 ℃
Detection : PDA 270 nm
Injection Volume : 0.5 μL
0 2 4 6 8 0 2 4 6 8 Mixer Volume : The volume of solvent delivered in 1 minute
Time (min) Time (min) Standard approx. 1.0 mL
Semi-micro approx. 0.4 mL
Low Low Sample；
Pressure 1 Pressure
1
2 3 1. 4-Methylphenol
Gradient 45 Gradient 2 3 4 5
2. 4-Ethylphenol
3. 4-Propylphenol
4. 4-Butylphenol
5. 4-Pentylphenol

0 2 4 6 8 0 2 4 6 8
Time (min) Time (min)

Gradient Retention (min) Gradient Retention (min)

Mode Peak 1 Peak 5 Mode Peak 1 Peak 5
23
High 3.16 5.76 High 3.19 5.90
Low 3.16 6.12 Low 3.19 6.83
(8-3) Adjusting the Sample Injection Volume
It is recommended to adjust the sample injection volume when scaling down the column to a
narrower internal diameter size.

●When the injection volume is the same, the sensitivity will increase when using a
concentration-dependent UV detector.
Column ID 4.6 mm Column ID 3.0 mm HPLC Conditions
(Flow Rate 1.0 mL/min) (Flow Rate 0.42 mL/min) Column : InertSustain C18
Standard 3 μm, 150 x 4.6 mm I.D.
1600 1600 5 Semi-micro 3 μm, 150 x 3.0 mm I.D.
Eluent : A) CH3CN
1400 1400 B) H2O
A/B = 65/35, v/v
1200 1200 Column Temp. : 40 ℃
2 Detection : UV 254 nm
1000 1000 4 Injection Volume : 10 μL
3
800 800 Sample；
1. Uracil
600 600 2. Acetophenone
2 5 3. Benzene
4. Toluene
400 400
3 4 1
5. Naphthalene

200 200
1
0 0
0 2 4 6 8 0 2 4 6 8
Time (min) Time (min)

●When scaling down the column to a narrower internal diameter size, but with the same
column length, the sample loading volume is generally proportional to the column’s cross-
sectional area. Therefore, deteriorated peak shapes may be observed when the polarity of
mobile phase and that of injection solvent are significantly different. In such case, the
injection volume should be reduced proportionally to the flow rate or the sample solution
should be diluted with the mobile phase.

Injection Volume 50 μL

Column ID 4.6 mm
(Flow Rate 1.0 mL/min)
5
4
2 3
mAU
1000

1
Injection Volume 10 μL
0

0 2 4 6 8
Time (min)

Column 2.1 mm
Column ID 2.1 mm (Flow Rate 0.2 mL/min)
(Flow Rate 0.2 mL/min)
2000

5 5
4
mAU

2 3
2000

4
1000

2
mAU

3
1000

1
1
0

0 2 4 6 8
0

Time (min)

0 2 4 6 8 10
Time (min)

24
(8-4) HPLC Tubing
It is necessary to select the appropriate tubing in your HPLC system depending on the internal
diameter size of an analytical column and flow rate of the mobile phase.
Extra-column volume and internal diameter of the tubing connecting the column outlet and
detector inlet can greatly impact instrument band spreading, which will effect the peak shape
and efficiency. It is recommended to decrease the internal diameter of tubing as it can decrease
band spreading. Make sure not to select the internal diameter of the tubing too narrow as it can
generate high back pressure.

●Effect of Extra-Column Volume on Chromatographic Separation

As shown below, larger extra-column volume will negatively impact the chromatographic
separation. Caution needs to be taken especially when using semi-micro HPLC systems.

Tubing ID 0.50 mm Tubing ID 0.25 mm Tubing ID 0.13 mm

Tubing Length 0.4 m Tubing Length 0.4 m Tubing Length 0.4 m

0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
Time (min) Time (min) Time (min)

Rs NA Rs 1.055 Rs 1.302
Efficiency NA Efficiency 3,908 Efficiency 5,673

HPLC Conditions
Column : InertSustain C18 HP (3 μm, 150 × 2.1 mm I.D.)
Eluent : CH3CN / H2O = 50/50, v/v
Flow Rate : 0.2 mL/min
Column Temperature : 40 ℃
Detection : UV 254 nm
Sample : o, p-Cresol

As shown below, the usage of narrower internal diameter tubing delivers better peaks even
under the same tubing volume.

Tubing ID 0.50 mm Tubing ID 0.25 mm

Tubing Length 0.2 m Tubing Length 0.8 m
Tubing Volume 39.25 μL Tubing Volume 39.25 μL

0 2 4 6 8 0 2 4 6 8
Time (min) Time (min)

25
Rs NA Rs 1.009
Efficiency NA Efficiency 3,765
(8-5) Detector Flow Cell Volume
When scaling down the column to a narrower internal diameter size, the sample diffusion
occurring not only at the tubing and the column, but at the detector flow cell can also influence
chromatographic results. When using narrower internal diameter columns, it is important to
use smaller volume flow cells.

●Comparison of Chromatographic Results using different Volumes of Detector Flow Cell

(Column ID 2.1 mm, Flow Rate at 0.2 mL/min)

Standard Flow Cell (Volume 12 μL) HPLC Conditions

Column : InertSustain C18
(2 μm, 150 x 2.1 mm I.D.)
5
Eluent : A) CH3CN
Efficiency of Peak 5: 6,940 B) H2O
Rs of Peak 4, 5: 3.13
2000

A/B = 65/35, v/v

4 Column Temp. : 40 ℃
2 Detection : PDA, 254 nm
mAU

3 Injection Volume : 10 μL
1000

Sample；
1
1. Uracil
2. Acetophenone
0

0 2 4 6 8 10 3. Benzene
Time (min) 4. Toluene
5. Naphthalene

Semi-Micro Flow Cell (Volume 2.5 μL)

Efficiency of Peak 5: 8,649

2000

Rs of Peak 4, 5: 3.50
5
mAU

4
1000

2
3

1
0

0 2 4 6 8 10
Time (min)

If the structure (e.g., optical path length) of the flow cell is the same, lower volume
flow cell will deliver better resolution, however, the sensitivity will decrease.

●Comparison of Efficiency between Column Internal Diameter Size and Flow Cell Volume

Column ID (mm) Standard Flow Cell Semi-Micro Flow Cell

2.1 6,940 8,649
3.0 10,137 10,713
4.6 19,231 19,195

* The HPLC system was optimized to be used for a 4.6 mm ID analytical column.
* The column length was 150 mm.
* The efficiency was determined by using Naphthalene under a specific analytical condition.

26
(9) Tips on Maximizing Efficiency on UHPLC Columns

In HPLC, if the particle size of the packing material is decreased, separation efficiency increases.
As shown in the following van Deemter plot, smaller particles provide lower HETP values and
these are achieved at a higher linear velocity, which a high throughput analysis can be operated
by using a higher flow rate.
However, some optimizations (e.g., tubing volume, data sampling rate) are required to fully
maximize the performance of UHPLC columns. Poor peak shapes or resolution may be observed
when using and connecting to unoptimized LC systems.

25
2µm
3µm
20 4µm
5µm

15
(um)
HETP*

10

0
0 1 2 3 4
Linear Velocity (mm/s)

* HETP : HETP is an acronym for the Height Equivalent to the

Theoretical Plate. It arises from the Plate Theory and is
numerically equal to the column length divided by
efficiency (the number of theoretical plates) of the column.

(9-1) Reduce Extra-Column Volume

●Tubing Volume
Select the appropriate tubing in your HPLC system depending on the internal diameter size of
an analytical column and flow rate of the mobile phase. For more details, please see section
(8-4).

●Detector Flow Cell Volume

The sample diffusion occurring at the detector flow cell can influence chromatographic results.
For more details, please see section (8-5).

(9-2) Data Sampling/Acquisition Rate

The data sampling rate determines how often a detector-signal data point is recorded to
construct the chromatogram. Generally, each peak should ideally be defined by at least 20 data
points. Insufficient data points may cause less separation efficiency or irreproducible
quantitation results. When using UHPLC columns, the sampling rate should be set less than 100
ms.
27
 Sampling Rate： 100 ms Sampling Rate ：1000 ms
Efficiency: 6782 Efficiency：5922

0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Time (min) Time (min)

Enlarged view
from 0 to 60
HPLC Conditions seconds
Column : InertSustain AQ-C18
(1.9 μm, 50 x 2.1 mm I.D.)
Eluent : A) CH3CN
B) H2O
A/B = 65/35, v/v

Column Temp. : 40 ℃
Detection : UV, 254 nm (Response: 0.1 sec)
Injection Volume : 0.2 μL

Sample；

1. Uracil
2. Acetophenone
3. Benzene
4. Toluene
5. Naphthalene
0.2 0.4 0.6 0.8 1.0
Time (min)

(9-3) Response Time/Time-Constant

Response time describes how fast the detector signal follows a sudden change of absorbance in
the flow cell. To achieve an optimal signal-to-noise ratio, the response time should be set three
times higher than the period of baseline noise. However, higher response time can produce
shorten and broaden peaks. As the peak widths are very narrow when using UHPLC columns,
the response time should be adjusted lower.

Response Time：0.05 sec Response Time：0.5 sec Response Time：1.5 sec

Efficiency：7137 Efficiency：5879 Efficiency：2993

1.0 1.0 1.0

HPLC Conditions
Column : InertSustain AQ-C18
(1.9 μm, 50 x 2.1 mm I.D.)
Eluent : A) CH3CN
B) H2O
A/B = 65/35, v/v

Column Temp. : 40 ℃
Detection : UV, 254 nm (Sampling Rate: 100 ms)
Injection Volume : 0.2 μL

Sample；

1. Uracil
2.
3.
Acetophenone
Benzene
28
4. Toluene
5. Naphthalene
(10) Benefits of Metal-Free PEEK Columns

(10-1) Benefits of Metal-Free PEEK Columns

Analytes containing phosphate groups creates the formation of phosphate-iron complexes,
which often lead to deteriorated peak shape or poor precision of quantitative result. In the past,
the residual metallic impurities in the packing material were assumed to be the cause of such
problem. However, the influence of the material of the column hardware also provides a big
impact to such trace analytes in high sensitivity analysis. Some improvement may be observed
when using mobile phases containing phosphate buffer or EDTA, however, these are not
compatible with LC/MS(/MS) applications.
The metal-free PEEK columns, including all wetted parts are made of polyetherether ketone
(PEEK), are available to improve chromatographic results with better peak shape and S/N for
bio-chromatographic applications.

Packing Material
Packing Material PEEK
Packing Material

SUS
PEEK PEEK

SUS

UHPLC-PEEK Columns PEEK Columns

(Available for 3 and Sub-2 μm particles) (Available for 4 to 10 μm particles)

●Comparison of Chromatographic Results between SUS and Metal-Free PEEK Columns

Analytes such as ATP contains phosphate groups, are highly chelating compound. As shown
below, Metal-Free PEEK column delivers improved peak shape and S/N compared to a SUS
hardware column.
4 4

3
3
1
2
2 2

2
1
0 0
0 2 4 6 8 0 2 4 6 8
Time (min) Time (min)

SUS Hardware Column Metal-Free PEEK Column

1. ATP HPLC Conditions

Column : InertSustain AQ-C18
2. ADP
(3 μm, 150 x 3.0 mm I.D.)
3. AMP
Eluent : 5 mM HCOONH4 in H2O
Column Temp. : 40 ℃
500 μg/L each
Detection : UV, 260 nm
29
Injection Volume : 10 μL
(10-2) Handling of Metal-Free PEEK Columns
Please note the handling of these PEEK columns are different from those traditional
metal stainless steel hardware columns. Please read the following before use.

●All the wetted parts are made of PEEK in UHPLC PEEK / PEEK columns. The structure of the
Column Port is especially delicate. Make sure not to over tighten the end-fittings. Over-
tightening the end-fittings will cause damage to the connection parts.

10-32UNF

●End-fittings are 1/16” Parker style, UP type.

2.4 m m

●The connection of PEEK columns can be done with less strength/force when comparing from
those traditional metal stainless steel hardware columns. Gently connect the PEEK columns to
the tubing and check if there are no leakage. The tightening strength/force of the torque is
approximately 0.8 N・m.

●Never use worn fittings. The usage of worn fittings will cause damage to the Column Port.
Make sure to always use new fittings.

●As shown below, when installing or removing the column, hold the end-fitting and end nut to
tighten or loosen the connection.

End Nut

Column Port

●When storing the column, use the provided column plugs and seal the column. Make sure not
to over tighten the column plugs when sealing the column. Over-tightening the column plugs
will cause damage to the connection parts.

●PEEK generally shows excellent solvent resistance to a wide range of organic solvents.
However, the use of THF or chloroform damage the surface of PEEK and cause irreparable
damage. Avoid the use of THF and chloroforms.

30
(11) Tips on Maximizing Purification Efficiency on Preparative Columns

The chemical properties of packing materials used for preparative separations must be chosen
not only to achieve the selectivity necessary for optimal separations but also the loadability that
enables maximum throughput. In this section, tips on considering the sample loadability and
selection of appropriate column dimensions are described.

(11-1) Sample Loadability

In preparative HPLC, the goal is to isolate and purify the target analyte. The peak shape of
target analyte may not look sharp, however, the resolution needs only to be maintained
between the peak of interest and the nearest peaks.

●Step ① : Evaluate the Sample Loadability

・Prepare a sample solution as concentrated as possible.

↓
・Inject the sample solution with small increments of injection volumes to evaluate the
sample loadability.
(To efficiently evaluate the sample loadability, use various injection volumes of 2 to 10
folds)
↓
・Once the injection volume increases, the peak shape will start deteriorating. The injection
volume where the peak shape deteriorated should be considered as the maximum sample
loadability.

Injection Vol.: 10 μL Injection Vol.: 50 μL Injection Vol.: 100 μL Injection Vol.: 250 μL
(Loadability 1.0 mg) (Loadability 5.0 mg) (Loadability 10 mg) (Loadability 25 mg)

3.0 4.0 5.0 6.0 3.0 4.0 5.0 6.0 3.0 4.0 5.0 6.0 3.0 4.0 5.0 6.0
Time (min) Time (min) Time (min) Time (min)

Column : Inertsil WP300 C18 (5 μm, 250 x 4.6 mm I.D.)

Eluent : CH3OH / H2O = 90/10, v/v
Flow Rat : 1.0 mL/min
Column Temp. : 40 ℃
Detection : UV 270 nm
Sample ：tert-Buthylbenzene (Appox. 10 ％, Diluent: Eluent)

As shown above, the peak shape started to be deteriorated when injecting 100 μL. The
peak shape was completely deteriorated and split into two peaks when injecting 250
μL. For this particular method and under this condition, we can determine the
maximum sample loadability is approximately 100 μL (10 mg).
31
●Step ② : Optimize Separation between the Target Analyte and nearest peaks

・ Prepare a sample solution as concentrated as possible.

↓
・Inject the sample solution with small increments of injection volumes to evaluate the
resolution.
(To efficiently evaluate the resolution, use various injection volumes of 2 to 10 folds)
↓
・Once the injection volume increases, the resolution is sacrificed as the column is in an
overload situation. Resolution factor larger than 1.5 is referred as complete baseline
separation between two neighboring peaks. Depending on the purification criteria,
Resolution factor around 1.2 may be acceptable.

Injection Vol.: 50 μL Injection Vol.: 100 μL Injection Vol.: 200 μL Injection Vol.: 400 μL
(Total Loadability 4.5 mg) (Total Loadability 9.0 mg) (Total Loadability 18 mg) (Total Loadability 25 mg)

0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Time (min) Time (min) Time (min) Time (min)

Rs 2.7 Rs 1.8 Rs 1.2 Rs 0.8

Column : InertSustain C18 (5 μm, 250 x 4.6 mm I.D.)

Eluent : CH3OH / H2O = 60/40 – 15 min – 100/0, v/v
Flow Rate : 1.0 mL/min
Column Temp. : 40 ℃
Detection : UV 270 nm
Sample : Alkylphenol C2-C4 (3.0 % each) Diluent: Eluent

If we were to define the Resolution factor of 1.2 as the limit of sample loadability,
we can determine the maximum injection volume is approximately 200 μL (total
loadability 18 mg) for this particular method and under this condition.

32
(11-2) Scale-Up
In the scale-up approach, a method developed for analytical purposes is directly applied to
larger internal diameter columns. The most important part is to understand the ratio of cross-
sectional area between the analytical and preparative column.

●Step ①：The flow Rate is Proportional to the Cross Sectional Area of the Column

The following HETP was plotted using three columns packed with 10 μm packing material
having different internal diameters. Lower the HETP, higher the efficiency.
Regardless of the column internal diameter size, the optimal linear velocity is at 3.0 cm/min
(0.5 mm/sec). As a conclusion, regardless of the column internal diameter size, when using a
column packed with a 10 μm particle size with the same bonded phase, the flow rate should be
adjusted to meet the linear velocity at 3.0 cm/min to maximize the efficiency of the column.
The linear velocity of the mobile phase is determined by dividing the mobile phase flow rate by
the cross-sectional area of the column. When changing the column to a different size, the
linear velocity should be kept constant. To keep linear velocity constant, the flow rate should
be adjusted in proportion to the column cross-sectional area, which is directly proportional to
the square of the ratio of column diameters.

* The optimal linear velocity is dependent on the particle size and

separation mode. Before scaling-up to a larger internal diameter
column, make sure to use the same bonded phase and particle size
in both analytical and preparative scale.

●Step ②：The Sample Loadability is Proportional to the Cross Sectional Area of the Column

The following was plotted using three columns having different internal diameters, which
demonstrates the relationship between sample loadability and efficiency. For example, the
sample loadability of each column are as follows when the efficiency is 2000.

ID 6 mm : Approx. 1 mg
ID 20 mm : Approx. 10 mg
ID 50 mm : Approx. 70 mg

As a conclusion, the injection volume should also be adjusted in proportion to the column cross-
sectional area to achieve equivalent separation efficiency.

33