System Dead Volume : Tubing Connections Sample Diluent

Influence of Dead Volume to the Peak Shape

The effect of Sample Diluent

Column Injector

2
Conditions

Column Size : Inertsil ODS-3 5μm, 150 x 3.0 mm I.D.

Eluent : A) CH3CN B) H2O Together, we can do more. The sample was dissolved using…
2 Conditions
80 100 120 140

1 A/B = 5/95
100% Acetonitrile Column Size : InertSustain AQ-C18 5μm, 150 x 4.6 mm I.D.
Flow Rate : 0.4 mL/min

Column Cleaning and Storage

3 Eluent : A) CH3CN B) H2O
Col. Temp : 40 ℃
1 4
mAU

Detection : UV 215 nm A/B = 10/90

60

Sample : 1.Methamidophos Flow Rate : 1.0 mL/min

40

2.Acephate 0 2 4 6 8 10 12 14 16 Col. Temp : 40 ℃

20

Time (min) Detection : UV 280 nm

0

0 2 4 6 8 10
Time (min) Sample : 1. 5-Hydroxymethyl-2-furaldehyde
1 2. 2-Furfural
2 3. 2-Acetylfuran
Eluent 4. 5-Methyl-2-furfural
3 Injection :10 µL
4
Column Injector

General Method of Washing for Reversed Phase Column General Method of Washing for Normal Phase Column
80 100 120 140

0 2 4 6 8 10 12 14 16
2 Time (min)
1

The organic solvent can be either Acetonitrile or Methanol

mAU

Dead Volume Normal-phase separations depend upon polar adsorptive interact ions, which the bonded phase
60

is polar and the mobile phase is non-polar. Polar analytes will be more strongly retained than
40
20

non-polar analytes in normal-phase chromatography. Clean the column with polar solvents to
0

remove highly polar contaminants.

0 2 4 6 8 10
Time (min)

Reversed Phase Reversed Phase Reversed Phase

General Analysis Ion-Pair Buffer
Hexane < Chloroform < Tetrahydrofuran < 2-Propanol < Ethanol
Weak solvents Strong solvents
5-10% Organic Solvent 20% Organic solvent The Buffering Effect
30-120min 30-60min

System Dead Volume ●Cleaning Inertsil SIL-100A, Inertsil SIL-150A, Inertsil WP 300 SIL, Inertsil NH2, Inertsil CN-3,
Acetic Acid Buffer pKa = 4.6 Phosphoric Acid Buffer pKa = 2.2、7.2、12.4
Inertsil Diol, InertSustain NH2 Columns
Tubing Connections from the Injector to Column 100% Organic Solvent
15-60min
50-100% Organic Solvent 100% Organic Solvent
Clean the column with ethanol or 2-propanol. Because alcohol solvents are quite viscous, CH3COOH CH3COO- H3PO4 H2PO4- HPO42- PO43-
15-60min 15-60min
adjust the flow rate to avoid excessive column back pressure. 100 100

Influence of Dead Volume to the Peak Shape

Mole fraction（％）

Mole fraction（％）
75 75

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

Storage Storage
50 50

5-50% Organic Solvent 5-50% Organic Solvent Storage

5-50% Organic Solvent Method Flow Rate 1 mL/min
I.D.(mm) 25 25
15min 15min 15min Method Mobile Phase n-hexane/2-propanaol/acetic acid = 90 /10/0.1
0.5 0.25 0.13 Step 1 : Clean the column with 100 % 2-propanol at 0 .2 mL/min for at least 60 minutes. 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH（-） pH（-）
N = 1224 N = 4351 N = 5682
Rs= － Rs= 1.138 Rs= 1.314
200
Equilibrate it with the mobile phase CH3COOH ⇔ CH3COO- + H+ H2PO4- Nearly 100%
Length(mm) from the Injector to column

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

N=－ N = 3908 N = 5673

Rs= － Rs= 1.055 Rs= 1.302 The maximum buffer capacity The minimum buffer capacity
400
General Method of Washing for HILIC Column
In case when the pH = pKa

0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
●When the mobile phase does not contain any buffered mobile phases or ion-pairing reagents Efficient buffering effect can be obtained when the pH is set within ± 1 the pKa.

Use high concentration organic solvent to remove the highly lipophilic contaminants. Increase
Time (min) Time (min) Time (min)

N=－ N = 3765 N = 5553

Rs= － Rs= 1.009 Rs= 1.281 the content of organic solvent up to 100%. Then, flush the column with 5 column volumes. Under HILIC mode, polar analytes are retained with high organic mobile phases. The following
800 When observing excessive back pressure, reduce and adjust the flow rate. describes the elution strength of solvents used in HILIC mode. 10 mM Acetic Acid Buffer (pH 4.5) 10 mM Phosphoric Acid Buffer (pH 4.5)
Acetic Acid or Sodium Acetate solutions Phosphoric Acid or Sodium Dihydrogen
were prepared to 10 mM each and adjusted Phosphate were prepared to 10 mM each
0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Example Column Dimensions 4.6 mm I.D. x 250 mm the pH at 4.5 and adjusted the pH at 4.5
Tetrahydrofuran < Acetonitrile < 2-Propanol < Ethanol < Methanol < Water 1
Time (min) Time (min) Time (min)
Conditions
N=－ N = 3227 N = 5598 Method Flow Rate 1 mL/min Column Size : InertSustain C18 5 μm, 150 x 4.6 mm I.D.
Rs= － Rs= 0.899 Rs= 1.273 Method Mobile Phase acetonitrile/water = 65/35 Weak solvents Strong solvents Eluent : A) CH3CN B) H2O A/B = 65/35, v/v
2
1250 2 1 Flow Rate : 0.4 mL/min
Col. Temp. : 40 ℃
Step 1 : Clean the column with 100 % acetonitrile at 1 mL/min for at least 30 minutes. ●Cleaning InertSustain Amide Columns Leading Detection : UV254 nm
1 2
OH
COOH
0 2 4 6 8 0 2 4 6 8 0 2 4 6 8

To avoid precipitating mobile phases buffers within the column, clean the column with a Sample : 1: Benzoic Acid
Time (min) Time (min) Time (min)

2: Benzyl Alcohol
Conditions
●When the mobile phase contain ion-pairing reagents water/organic solvent mixture, using the same content as in the buffered mobile phase. Clean the
Column Size : InertSusatin C18 3 μm, 150 x 2.1 mm I.D. 0 2 4 6
Depending on the ion-pairing reagent type, precipitation may occur when cleaning the column with column with acetonitrile/water = 50/50 to remove highly polar contaminants. 0 2 4 6 Time (min)
Eluent : A) CH3CN B) H2O Time (min)
A/B = 50/50
100 % water and extreme care is required. Clean the column with a water/organic solvent mixture, If the column still shows shift in retention time or distorted peak shapes, clean the column with
Flow Rate : 0.2 mL/min using the same content as in the mobile phase containing an ion-pairing reagent. For example, 100 % water for at least 30 minutes. After cleaning the column, make sure to thoroughly
Col. Temp : 40 ℃
clean the column with 10% acetonitrile in water for at least 30 minutes. Then, clean it with equilibrate the column with the mobile phase to be used in the analysis prior to use. Store the
Detection : UV 254 nm
Sample : o,p-cresol
water/acetonitrile = 50/50 for at least 30 minutes. InertSustain Amide column in 100% acetonitrile.
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 Dimensions 4.6 mm I.D. x 250 mm
Method Flow Rate 1 mL/min
Example Column Dimensions 4.6 mm I.D. x 250 mm Method Mobile Phase 5 mM CH3COONH 4/acetonitrile = 10/90
Method Flow Rate 1 mL/min Step 1 : Clean the column with acetonitrile/water = 90/10 at 1 mL/min for at least 30 minutes.
Method Mobile Phase 10 mM KH 2PO 4+ 2 mM *IPCC-09 (pH:2.5)/acetonitrile = 90/10 Step 2 : Clean the column with acetonitrile/water = 50/50 at 1 mL/min for at least 30 minutes.
Gradient Delay 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. ●Cleaning Inertsil HILIC Columns
* IPCC-09 ：Sodium 1-nonanesulfonate To avoid precipitating mobile phases buffers within the column, clean the column with a
Low Pressure High Pressure * Please be aware that removal of 100% of the ion-pairing reagent may not be possible. Due to the water/organic solvent mixture, using the same content as in the buffered mobile phase. Clean Column Contamination
Gradient Mixing Gradient Mixing fact that ion-pairing reagents can alter column selectivity, it is strongly recommended to dedicate the column with 100 % water to remove highly polar contaminants.
System System columns to ion-pairing methods to avoid problems with reproducibility. Example Column Dimensions 4.6 mm I.D. x 250 mm
Mixing If your column is contaminated… Conditions
point of
solvents Method Flow Rate 1 mL/min Column Size : Inertsil ODS-3

Mixing
Method Mobile Phase 5 mM CH3COONH 4/acetonitrile = 10/90 3 μm, 100 x 3.0 mm I.D.
point of
solvents
●When the mobile phase contain buffered mobile phases Step 1 : Clean the column with acetonitrile/water = 90/10 at 1 mL/min for at least 30 minutes. Eluent : A) CH3CN B) H2O

Clean the column with a water/organic solvent mixture, using the same content as in th Step 2 : Clean the column with 100 % water in water at 1 mL/min for at least 30 minutes.
A/B = 65/35, v/v
Flow Rate : 0.4 mL/min
buffered mobile phase. For example, clean the column with 20% acetonitrile in water for at Col. Temp. : 40 ℃
A B C D A B C least 30 minutes. Then, clean it with 100 % acetonitrile. Detection : UV254 nm
Sample : 1: Acetophenone
●Cleaning InertSustain NH2 Columns 2: Benzene
Example Column Dimensions 4.6 mm I.D. x 250 mm To avoid precipitating mobile phases buffers within the column, clean the column with a 3: Toluene
Method Flow Rate 1 mL/min water/organic solvent mixture, using the same content as in the buffered mobile phase. Clean 0 2 4 6 0 2 4 6 4: Naphthalene

Method Mobile Phase 10 mM KH 2PO 4/acetonitrile ＝80/20 the column with acetonitrile/water = 50/50 to remove highly polar contaminants. Back pressure 14MPa Back pressure 8MPa
Column ID 4.6 mm Column ID 3.0 mm Conditions
（ Flow Rate 1.0 mL/min） （Flow Rate 0.42 mL/min）
Column Size : InertSustainSwift C18
Step 1 : Clean the column with 20% acetonitrile in water at 1 mL/min for at least 30 minutes. If the column still shows shift in retention time or distorted peak shapes, clean the column
High
Pressure
1
2
34
High
Pressure
12
3 4 5 Standard :3 μm, 150 x 4.6 mm I.D. Step 2 : Clean the column with 100% acetonitrile at 1 mL/min for at least 30 minutes. with 50 mM ammonium formate (or ammonium acetate) aqueous solution/acetonitrile =
Gradient Gradient
5
Semi-micro:3 μm, 150 x 3.0 mm I.D. * When using the column again for the analysis, follow the procedures below to avoid 50/50 for at least 30 minutes. After cleaning the column, make sure t o thoroughly equilibrate
Eluent : A) CH3CN B) H2O precipitating mobile phase buffers on the column. the column with the mobile phase to be used in the analysis prior to use. Store the
0 2 4 6 8
Step 1 : Equilibrate the column with 20% acetonitrile in water at 1 mL/min for at least 30
0 2 4 6 8
A/B = 50/50 – 6min – 100/0, v/v
InertSustain NH2 column in 100% acetonitrile.
Time (min) Time (min)
Col. Temp : 40 ℃
Low 1
2 3 Low
1 Detection : PDA 270 nm
minutes.
GL Sciences, Inc. Japan
2 3 4 5
Pressure
Gradient
4 5 Pressure
Gradient
Sample : o,p-cresol Step 2 : Equilibrate the column with the buffered mobile phase to be used at 1 mL/min for at Example Column Dimensions 4.6 mm I.D. x 250 mm
Mixer Volume:Standard approx. 1.0 mL
least 30 minutes. Method Flow Rate 1 mL/min
Semi-micro approx. 0.4 mL
Step 3 : The column may be considered fully equilibrated once a constant back pressure and 22-1 Nishishinjuku 6-Chome
0 2 4 6 8 0 2 4 6 8
Sample :1. 4-Methylphenol Method Mobile Phase 5 mM CH3COONH4/acetonitrile = 10/90
Time (min) Time (min)
stable baseline are observed. Shinjuku-ku, Tokyo,
Retention (min) Retention (min)
2. 4-Ethylphenol
Step 1 : Clean the column with acetonitrile/water = 90/10 at 1 mL/min for at least 30 minutes.
Gradient
Node
Gradient
3. 4-Propylphenol 163-1130, Japan
Peak 1 Peak 5 Node Peak 1 Peak 5
Step 2 : Clean the column with acetonitrile/water = 50/50 at 1 mL/min for at least 30 minutes.
High
Low
3.16
3.16
5.76
6.12
High 3.19 5.90 4. 4-Butylphenol
Phone: +81-3-5323-6620
Low 3.19 6.83 5. 4-Pentylphenol
Fax: +81-3-5323-6621
Email: world@gls.co.jp
Web: www.glsciences.com