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(HPLC) is an instrument that commonly used to carry out liquid chromatography to separate
constituent in a sample using a mixture of solvent (mobile phase) and a column of material
(stationary phase). Through this separation the signal of the constituents is manipulated for
chemical profiling or quantitative analysis of constituents of interest.. Because of this
versatility, HPLC is used in a variety of industrial and scientific applications, such as
aspharmaceutical, environment, forensics and chemical.

Sample retention time will vary depending on the interaction between the stationary phase,
the molecules being analyzed, and the solvent, or solvents used. As the sample passes through
the column it interacts between the two phases at different rate, primarily due to different
polarities in the analytes. Analytes that have the least amount of interaction with the
stationary phase or the most amount of interaction with the mobile phase will exit the column


1. The sample is placed into three replicates.

2. After that, 5ml of methanol is added into the sample.

3. The sample is sonicated for 15 minutes. After that, the sample is filtered through 0.45m
syringe filter and placed it into the vials.

4. The sample is labeled.

5. Then, the sample is ran by using HPLC instrument.


Figure 1 Total yield of extract and cost efficiency for sequential of number of extraction steps

Figure 2 : Amount of eurycomanone for multi-steps extractions

Figure 3 : Amount of active ingredients for multi-steps extractions


While distillation works on the principle of boiling point difference, liquid-liquid extraction
works on the principle of chemical structure difference. This makes extraction ideally suited
for separation problems such as those listed below.

Fermentation and Algae Broths

Biofuels and chemicals produced by biological processes such as fermentation and
algae often require liquid-liquid extraction (LLE) as the first step in recovery and

Phenol from Wastewater

Removal of high boiling organics from wastewater; such as phenol, aniline and
nitrated aromatics.

Acetic Acid Extraction

Recovery of tightly hydrogen-bonded organics from water; such as formaldehyde,
formic acid and acetic acid.

Essential Oil Extraction

Essential oil extraction; such as pharmaceuticals, flavors, fragrances and food

Caprolactam Extraction
Recovery of products from reactions; such as caprolactam and adiponitrile (for nylon
production), acrylic acids and agricultural chemicals.

Neutralization/washing of acids or bases from organic stream

Such as acrylates, nitrated organics and chloro-benzene compounds.


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