Unlocking the Potential of Derivatization Technology in HPLC Analysis
In modern analytical chemistry, laboratory analysts frequently encounter challenges when certain compounds resist detection or separation using conventional methods. This is where derivatization technology steps in—a powerful tool designed to enhance detection sensitivity and separation performance.
This article delves into the application of derivatization techniques in high-performance liquid chromatography (HPLC) and their role in overcoming analytical obstacles.
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What is Derivatization?
Derivatization involves modifying analytes through chemical reactions with specific reagents, introducing functional groups that improve their detectability or separability.
Widely used across various instrumental analysis methods—including chromatography (gas, liquid, supercritical, thin-layer, and capillary electrophoresis), mass spectrometry, NMR spectroscopy, UV-Vis, fluorescence, and electrochemical analysis—this technique has become a cornerstone in solving analytical challenges.
Derivatization in Gas Chromatography vs. Liquid Chromatography
In gas chromatography (GC), derivatization enhances sample volatility and detection sensitivity. Conversely, in HPLC, derivatization typically employs reagents (known as derivatizing or labeling reagents) to react with target compounds, forming products that are more detectable or separable.
Types of Derivatization in HPLC
HPLC derivatization can address various issues, such as poor retention, instability, and low sensitivity, by converting analytes into easily detectable derivatives. This process can be categorized by the nature of the reaction, reaction location, and integration with the analytical instrument:
By Bond Formation:
- Labeled Reactions: Form covalent bonds between the analyte and the derivatizing reagent.
- Non-Labeled Reactions: Involve non-covalent interactions, such as ion pairing, photolysis, oxidation-reduction, or electrochemical reactions.
By Reaction Location:
- Pre-Column Derivatization: Occurs before the sample enters the chromatographic column.
- On-Column Derivatization: Takes place within the column.
- Post-Column Derivatization: Happens after chromatographic separation.
By Instrumentation Integration:
- Online Derivatization: Fully automated and integrated with the instrument.
- Offline Derivatization: Conducted manually before the analytical process.
- At-line Derivatization: Semi-automated, involving dedicated equipment for complex derivatization workflows.
Common Reactions in HPLC Derivatization
The versatility of HPLC derivatization is evident in the variety of chemical reactions it employs, including esterification, acylation, alkylation, silylation, boronation, cyclization, ionization, and photochemical reactions. The choice of reaction depends on the analyte’s characteristics and the analytical goals.
Pre-Column Derivatization
Pre-column derivatization transforms the analyte into a detectable derivative prior to chromatographic separation. This method can be executed online or offline.
- If online, the analyte and derivatizing reagent are injected through separate pumps and combined in real-time in reactors.
- If offline, the reaction of analyte and reagent are conducted first, and the resultant derivative is injected into the HPLC system.
- Another online method involves incorporating derivatizing agents into the mobile phase to directly interact with the analyte.
Post-Column Derivatization
Post-column derivatization involves modifying analytes after their separation in the HPLC column to improve their detectability. In this method, the separated compounds react with derivatization reagents before reaching the detector. This method is further categorized to UV-Vis, fluorescence, Raman, electrochemical, or photochemical derivatizations.
Ultraviolet-Visible (UV-Vis) Derivatization
UV derivatization involves reacting organic compounds of weak or no UV absorption with derivatization reagents containing UV-absorbing groups, generating UV-detectable compounds. For instance, amines can react with halogenated hydrocarbons, carbonyl, or acyl derivatives to produce such detectable derivatives.
Visible derivatization serves two main purposes:
- Transition Metal Ion Detection: Transition metal ions react with chromogenic reagents to form colored complexes, chelates, or ion-associated compounds, which are then detectable using visible light.
- Organic Ion Detection: By adding counterions of the analyte ion to the mobile phase, colored ion-pair compounds are formed. These compounds can be separated and detected via visible light techniques.
Fluorescence Derivatization
Fluorescence derivatization entails reacting the analyte with a fluorescence derivatization reagent to form a fluorescent compound for detection. Some fluorescence reagents themselves lack fluorescence but produce highly fluorescent derivatives.
While derivatization enhances sensitivity and selectivity, it also increases the complexity of the system, consumes additional time, and raises analytical costs. Moreover, certain derivatization reactions require strict control over reaction conditions. Thus it is considered only when no convenient, sensitive detection method is available or when enhancement to selectivity is required.
Photochemical Derivatization
Photochemical derivatization (PCD) is a method relying on photochemical reactions and combines with other detection methods to enhance sensitivity and selectivity. Fluorescence is the primarily combined method, and electrochemical is also often included.
PCD broadens the application scope of traditional detection methods and finds widespread use in analyzing pharmaceuticals, complex biological samples, and environmental samples.
Key photochemical reactions include intramolecular energy transfer, collisional energy transfer, quenching, photoionization, isomerization, direct reactions, and intermolecular decomposition.
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Applications of HPLC Derivatization
1. Aflatoxin Detection (Photochemical Derivatization)
In this application, photochemical derivatization enables low-concentration aflatoxin detection with fluorescence detectors, achieving higher sensitivity than conventional methods.
2. Amino Acid Analysis in Feed (Pre-Column Derivatization)
Column: Welch Ultisil Amino Acid (4.6×250 mm, 5 μm)
3. Formaldehyde in Cosmetics (Post-Column Derivatization)
Applicable to various cosmetic forms, namely water-based liquid, cream and lotion, gel, oil-based liquid, wax-based, and powder.
Based on the detector in use, one of the two condition sets below is chosen:
Conditions for PDAD detector
| Column |
C18 column (4.6mm×250mm, 5μm) or equivalent |
| Mobile phase |
Phosphoric acid solution |
| Flow rate |
1.0 mL/min |
| Column temperature |
20 ℃ |
| Detector wavelength |
420 nm |
| Injection volume |
10 μL |
Conditions for FLD detector
| Column |
C18 column (4.6mm×250mm, 5μm) or equivalent |
| Mobile phase |
Phosphoric acid solution |
| Flow rate |
1.0 mL/min |
| Column temperature |
20 ℃ |
| Excitation wavelength |
425 nm |
| Emission wavelength |
510 nm |
| Injection volume |
Conditions of post-column derivatization
| Derivatization solution flow rate |
0.8 mL/min |
| Reactor temperature |
100 ℃ |
4. Taurine in Eye Drops (Post-Column Derivatization)
Preparation of test solution:
Accurately measure 3 mL of the sample and transfer it into a 50 mL volumetric flask. Dilute to volume with water and mix well. Then, accurately measure 5 mL of the solution and transfer it into a 100 mL volumetric flask. Dilute to volume with water and mix well.
Preparation of reference solution:
Accurately weigh an appropriate amount of taurine reference standard. Dissolve and dilute with water to prepare a solution with a concentration of approximately 0.15 mg/mL.
Chromatographic conditions:
| Column |
C18 column |
| Mobile phase |
Phosphate buffer solution (pH 7.0) / acetonitrile / water = 70:15:15 |
| Column temperature |
60 ℃ |
| Detector wavelength |
338 nm |
Post-Column Derivatization Method:
| Derivatization Reagent |
Prepare an ortho-phthalaldehyde (OPA) solution as follows: Dissolve 24 g of sodium hydroxide and 43.2 g of boric acid in approximately 2700 mL of water. Adjust the pH to 4.0 with sulfuric acid, add 2 mL of 2-mercaptoethanol and 10 mL of 8% ethanol solution of OPA, and dilute to 3000 mL with water (prepare freshly before use). |
| PTFE Reaction Tube |
Use a reaction tube with an inner diameter of 1 mm and a length of approximately 40 cm. |
| Derivatization Temperature |
60 °C |
| Derivatization Reagent Flow Rate |
0.5 mL/min |
| Injection volume |
10 μL |
