LC Technical Note

LT133

GL Sciences Inc.

How to use preparative HPLC - Part 1
Evaluation of loading amountIn preparative HPLC, it is quite important to investigate loading amount per one injection because required
times of injection and suitable column dimension can be estimated from loading amount.
In this note, how to evaluate loading amount using analytical column (4.6 mm I.D.) is described.
(K. Kanno)

Conditions of preparative HPLC are generally optimized as the following flowchart.

Find out appropriate
separation conditions using
analytical column-Composition of mobile phase-Gradient programetc.

Evaluate loading amountusing analytical column

Described in this note

Scaling up to preparativecolumn

Described in LC Technical
Note No. 134.

Preparative HPLC

＜What is important to evaluate loading amount＞
Main purpose of preparative HPLC is not quantitation but purification of sample. Therefore, good peak shape is
not necessarily required. It dose not matter , either, if injected amount exceeds detector’s upper limit oflinearity. Following two points are important to evaluate loading amount;1)All amount of the target compound is well retained on column
2)Target compound is successfully separated from other peaks

GL Sciences LC Technical Note1) All amount of the target compound is well retained on column① Prepare sample solution at as high concentration as possible.
② Begin with small amount, and then increase injection volume step-by-step.
In the figure shown below, injection volume was changed as follows; 10 µL → 50 µL → 100 µL → 250 µL. It
should be efficient to evaluate peak shape every time increasing injection volume from 2 to 10 times.
③ Peak shape gets worse as injection volume is increased. Injection volume at which peak shape begins to bedeteriorated is the maximum amount under the conditions.
In the figure shown below, the peak began to lose its shape when 100 µL of sample solution was injected. Peakwas split into two with 250 µL injection. Therefore, it can be said that upper limit of injection volume (amount)under the condition should be around 100 µL (10 mg).

Inj. Vol.：50 µL(5.0 mg)

Inj. Vol：10 µL(1.0 mg)

3.0

4.0
5.0
Time (min)6.

ColumnEluent Flowrate
Temperature
Detection
Sample3.0

4.0
5.0Time (min)6.

Inj. Vol.：250 µL(25 mg)Inj. Vol. ：100 µL(10 mg)

3.0

4.0
5.0
Time (min)

6.

3.0

4.0
5.0
Time (min)6.

: Inertsil WP300 C18 (5 μm, 250 x 4.6 mm I.D.): CH3OH/H2O = 90/10, v/v (gradient mixer): 1.0 mL/min: 40 ℃: UV 270 nm：tert-Buthylbenzene (approx. 100 mg/mL) dissolved in eluent

In case the maximum injection volume obtained with 4.6-mm I.D. column is 100 µL as the figure shown
above, the maximum injection volume of preparative column is estimated as follows. Detail of scaling up
is described in LC Technical Note No.134.

Inner diameter (I.D.) of column
Relative cross-sectional area
The maximum injection volume4.6 mm
1100 µL

10 mm
4.7470 µL

14 mm
9.3930 µL

20 mm
18.9
1.89 mL

30 mm
42.5
4.25 mLGL Sciences LC Technical Note
2) Target compound is successfully separated from other peaks①Prepare sample solution at as high concentration as possible.
② Begin with small amount, and then increase injection volume step-by-step.
In the figure shown below, injection volume was changed as follows; 50 µL → 100 µL → 200 µL → 400 µL. It
should be efficient to evaluate separation of compounds every time increasing injection volume from 2 times
to 10 times.
③ The separation gets worse as injection volume is increased. Peak resolution (Rs) is quite useful to evaluate
whether sufficient separation is achieved. Target value of Rs varies depending on required purity or how to
fractionate the peak, while baseline separation (Rs > 1.5) is ideal. Rs value of between 1.2 and 1.5 is often
sufficient when fractionation is carried by detecting peak height or peak slope.
In the figure shown below, it can be said that upper limit of injection volume (total loading amount) should bearound 200 µL (18 mg) in case the target value of Rs is set at 1.2.

Inj. Vol.：50 µL

Inj. Vol.：100 µL

Inj. Vol.：200 µL

Inj. Vol.：400 µL(Total load： 4.5 mg)(Total load： 9.0 mg)(Total load：18 mg)(Total load：36 mg)

Rs = 2.7

Rs = 1.80 2 4 6 8 10 12 14Time (min)0 2ColumnEluent
Flow rate
Temperature
DetectionSample

4 6 8 10 12 14Time (min)Rs = 1.2

Rs = 0.80 20 24

6 8 10 12 14Time (min)

4 6 8 10 12 14Time (min): InertSustain C18 (5 μm, 250 x 4.6 mm I.D.): CH3OH/H2O = 60/40 – 15 min – 100/0,v/v: 1.0 mL/min: 40 ℃: UV 270 nm：Alkylphenols (C2-C4, 30 mg/mL each) dissolved in eluent

In case the maximum injection volume obtained with 4.6 mm I.D. column is 200 µL as the figure shown above, themaximum injection volume of preparative column is estimated as follows. Detail of scaling up is described in LC
Technical Note No.134.
Inner diameter (I.D.) of column
Relative cross-sectional area
The maximum injection volume4.6 mm
1200 µL

10 mm
4.7940 µL

14 mm
9.3
1.86 mL

20 mm
18.9
3.78 mL

30 mm
42.5
8.5 mLGL 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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