LC Technical Note

Improvement of purification using HPLC column～ Comparison between column chromatography andpreparative HPLC ～LT114
GL Sciences Inc.

To purify crude product of organic synthesis or to isolate

optimal flow rate for 5 µm - 10 µm particle is faster thansome compounds from natural product, columnthat for 50 µm - 200 µm particle.

chromatography with silica gel of 50 µm - 200 µm particle is

We recommend purification using HPLC when sufficient

generally used. However, separation efficiency can beperformance is not offered with conventional column

improved by use of 5 µm - 10 µm particle for HPLC column.

chromatography or flash column chromatography. （C.

Moreover, separating time can be reduced becauseAoyama）

Conditions;

Separation using 50 μm particleColumn（Flow rate: 0.1 mL/min）
1Eluent

2 3Column Temperature
Detection

Injection Volume0100300
300

200
Time [min]: ODS column (250×4.6mm I.D.): A) CH3CNB) 0.1% HCOOH in H2O A/B= 40/60, v/v: 40 ℃: UV 270 nm: 10 μL

1. sec-Butyl p-Hydroxybenzoate
2. iso-Butyl p-Hydroxybenzoate
3. n-Butyl p-Hydroxybenzoate

Even when separation is difficult
with conventional column
chromatography, …

Separation using 5 μm particle（Flow rate: 1.0 mL/min）
1

By use of 5 μm particle,
baseline separation was achieved !
and run time is also reduced !

23If flow rate was increased as theparticle size was unchanged, …

010203030

Time [min]Separation with 50 μm particle and 1.0 mL/min
12+3

Separation got worse because the flow rate was shiftedfrom optimal condition.

01020
Time (min)In general, optimal flow rate bears an inverse
relation to particle size.

30Particle size [µm] Typical flow rate* [mL/min]

235102050

2.5
1.7
1.00.50.20.1* Optimal value for ODS column of 4.6 mm
inner diameter.

GL Sciences LC Technical Note
An Example of Scale-up using HPLC column
If inner diameter of HPLC column is changed to another, almost the same elution order and retention time should beobtained by adjusting flow rate depending on the inner diameter. Therefore, solvent usage and sample amount can besaved by using columns of small inner diameter for investigation of HPLC condition. (Detail information about relation
between inner diameter of column and flow rate is described in LC technical note No.87.)

Conditions;
ColumnEluent: Inertsil ODS-3 （5 μm, 250 mm length）: A) CH3CNB) 0.1 % CF3COOH in H2O
A/B = 40/60, v/v: 40 ℃: UV 270 nm: Glycyrrhizae radix extract (2.0 mg/mL)

Temp.
Detection
SampleBy switching inner diameter of column,
20 times increase of loading amount wasachieved without changing the elution order.

1. Glycyrrhizic acid10246810Time (min)11214

Inner diameter: 4.6 mm
Flow rate: 1.0 mL/min
Injection volume : 160 μL
Loading amount : 0.32 mg

160246810Time (min)Inner diameter
Flow rate
Injection volume
Loading amount121416: 20 mm: 18.9 mL/min: 3.0 mL
: 6.0 mg

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly. We reserve theright to amend this information or data at any time and without any prior announcement.

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