Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique

Summary

The present protocol developed a method to estimate the yield of compounds on the TLC plate using the blue-LED illumination technique. The advantages of this approach are that it is safe, effective, inexpensive, and allows the researcher to measure multiple samples simultaneously.

Abstract

Thin-layer chromatography (TLC) is an accessible analytical technique that has been extensively used in organic chemistry research to quantify the yield of unknown samples. The present study developed an effective, cheap, and safe method to estimate the yield of samples on a TLC plate using the blue-LED illuminator. Lovastatin extracted from Aspergillus terreus was the example compound used in the present study. Regression models based on the lovastatin standard were used to evaluate the yield of lovastatin. Three methods were compared: bioassay, UV detection, and blue-LED illumination. The result showed that the blue-LED illumination method is significantly more time-effective than UV detection and bioassay methods. Additionally, the blue-LED illumination was a relatively safe option because of the concern of biological hazards in the bioassay method (e.g., microbial infection) and ultraviolet exposure in the UV detection method. Compared to the expensive methods requiring specialized instruments and long-term training before working independently, such as GC, HPLC, and HPTLC, using the blue-LED illuminator was an economical option to estimate the yield of samples from a TLC plate.

Introduction

Thin-layer chromatography (TLC) is widely used as a qualitative and quantitative technique in the field of organic chemistry^1,2,3. The main advantages of TLC are that it provides fast detection, flexible sample requirements, and does not require specialized equipment⁴. To date, even though many advanced approaches have been established, TLC is still the main method for identifying unknown samples in a mixture. However, the challenge of this approach is the lack of safe and inexpensive equipment for quantifying the sample yield, especially for developing laboratories with limited budgets. The present study, therefore, aimed to develop an efficient, safe, and inexpensive method combining with TLC to estimate the yield of the samples.

Unlike high-performance TLC (HPTLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC) with strict sample requirements, time-consuming, and involvement of multistep for sample preparation^1,5, TLC showed several advantages. First, for sample preparation, the HPLC and GC cannot detect the crude extract because the crude extract may plug the column of HPLC and GC. Second, when the samples are not UV-suitable (important for HPLC analysis) or with low volatility (important for GC analysis), TLC can be applied to these samples, and the use of visualization reagent makes the isolated samples visible on thin layers^6,7,8. Third, for general users, HPLC and GC generally require a relatively long time pre-training before working independently, compared to TLC. In addition, quantitative TLC analysis, known as high-performance TLC (HPTLC), can digitize the information on a TLC plate with a highly sensitive scanner. However, the cost of the HPTLC system is relatively expensive. As such, developing a cost-effective and fast approach to quantify samples on the TLC plate is an important topic.

Similar methods have been developed for TLC yield quantification; for example, Johnson⁹ reported a technique that allows the quantification of the samples on a TLC plate by using a flatbed scanner attached to a computer. In 2001, El-Gindy et al.¹⁰ developed the TLC- densitometric method, which was used to detect the compound with optical density, and the technique was also applied by Elkady et al.¹¹. In 2007, Hess² presented the digitally enhanced-TLC (DE-TLC) method applied to detect the yield of a compound on a TLC plate using a digital camera combined with UV light. Hess also compared the cost differences between HPTLC and DE-TLC method and concluded that the DE-TLC method could be used in high school and college labs because of its affordable cost². However, the cost of the TLC-densitometric method was still expensive, and the operation of ultraviolet light requires adequate pre-training in case the users might get exposed to ultraviolet radiation. Therefore, compatible with TLC, developing an efficient, safe, and inexpensive method to quantify the sample yield is desirable.

The present study described a protocol for detecting the sample on a TLC plate using the blue-LED illuminator, and developed a regression model with high reliability (high R-square value) to measure the dimensions of the bands, and then determine the compound yield. Finally, it was found that the blue-LED illumination method is a relatively safe (vs. UV-detection method), cheap (vs. GC, HPLC, and HPTLC), and effective (vs. bioassay method) approach for yield quantification.

Protocol

The present protocol is described using lovastatin as an example. Lovastatin was extracted from one-week-old Aspergillus terreus.

1. Compound extraction

NOTE: For details on compound extraction, please see Figure 1.

1. Culture Aspergillus terreus on the potato dextrose agar (PDA, see Table of Materials) medium at 30 °C.
2. Dry the culture at 40 °C for 24 h. Transfer the dried culture into a 50 mL tube using sterilized tweezers and add 15 mL ethyl acetate.
3. Shake the mixture vigorously by vortexing for 1 min and incubate for 1 h at 40 °C with shaking at 200 rpm.
4. Sonicate the mixture using a 40 kHz ultrasonic bath (see Table of Materials) at 40 °C for 1 h.
5. Centrifuge the mixture at 5,000 x g for 1 min at room temperature and filter through an 11 µm filter paper.
6. Extract the filtrate with an equal volume of sterile water in a separatory funnel.
7. After phase separation, collect the organic layer, and then evaporate in a rotary evaporator (see Table of Materials). Dissolve the residue in 2 mL of ethyl acetate.

2. Separation of the crude extract by normal phase (NP) adsorption column

1. Pack the column with NP silica gel as the stationary phase, and use n-hexane:ethyl acetate:trifluoroacetic acid (H:E:T; 80:20:0.1, v/v/v) as the mobile phase.
2. Load 2 mL of the extract (step 1) onto the column and add the mobile phase solvent at a flow rate of 1 mL/min to elute the extract.
   NOTE: The flow rate was manually controlled using a stopcock.
3. Verify the effluent by TLC to confirm the presence of lovastatin, and then evaporate in a rotary evaporator at 45 °C until the solvent gets removed. This step takes approximately 20-25 min.
4. Dissolve the residue in 1 mL of ethyl acetate, and then mix with an equal volume of 1% trifluoroacetic acid.
5. Centrifuge the mixture at 5,000 x g for 1 min at room temperature and collect the organic layer in a new glass tube.

3. Preparation and loading of thin-layer chromatogram (TLC) plates

1. Spot 5 µL of samples and lovastatin standards (see Table of Materials) onto the baseline of the TLC plate using a capillary pipette, leaving a border of 1 cm on the sides of the TLC plate.
2. Dry the TLC plate in a fume hood for 5 min at room temperature.
3. Place the plate gently by forceps in a saturated glass chamber containing the mobile phase solvent. Cover the chamber with a glass lid and allow the plate to develop fully.
4. Remove the plate from the chamber when the solvent line reaches 1 cm from the top of the plate.

4. Analysis by the blue-LED illuminator

1. Mark the solvent line with a pencil. Dry the plate in the fume hood for 10 min at room temperature.
2. After drying, immediately soak the plate in 10% H₂SO₄ solvent, and then dry in the fume hood for 10 min at room temperature.
3. Place the plate on the heating panel until the brown spots appear. Ensure that the plate is not overheated, as this might make visualization of lovastatin difficult.
4. Transfer the plate to the blue-LED illuminator and scan using a compatible freeware (MiBio Fluo) (see Table of Materials).

5. Yield estimation by the regression model

1. Measure the dimension of bands using the ImageJ software (see Table of Materials).
2. Establish a regression model using data analysis and graphing software (see Table of Materials) based on the descending concentrations of lovastatin standards, including 1 mg/mL, 0.75 mg/mL, 0.5 mg/mL, and 0.25 mg/mL.
3. Apply the regression model to estimate the yield of the samples.

Results

This study presented the blue-LED illumination method to estimate the yield of compounds, and this method was validated and compared to bioassay and UV-detected methods (Table 1). The regression models were developed based on the dimensions of bands and concentration of standards for three methods, respectively, to predict the yield of samples. First, in the results of the bioassay method, the R-square between the dimensions of the inhibition zone and lovastatin standards was 0.99, and the sample yield w...

Discussion

The present study described a new approach, the blue-LED illuminator, to quantify compounds without using expensive and specialized equipment, such as HPTLC, HPLC, and GC method, and the method was compared with the bioassay and UV-detected methods to evaluate quantification performance. As a result, it was concluded that the blue-LED illumination method is a relatively safe and effective protocol used to quantify the yield of targeted compounds on the TLC plate.

Previous studies have reported...

Materials

Name	Company	Catalog Number	Comments
American bacteriological Agar	Condalab	1802.00
Aspergillus terreus		ATCC 20542
Blue-LED illuminator	MICROTEK	Bio-1000F
Centrifuge	Thermo Scientific	HERAEUS Megafuge 8
Compact UV lamp	UVP	UVGL-25
Ethyl Acetate	MACRON	MA-H078-10
Filter Paper 125mm	ADVANTEC	60311102
ImageJ	NIH	Freeware	https://imagej.nih.gov/ij/download.html
Lovastatin standard	ACROS	A0404262
MiBio Fluo	MICROTEK	V1.04
n-Hexane	C-ECHO	HH3102-000000-72EC
OriginPro	OriginLab	9.1	https://www.originlab.com/origin
Potato dextrose broth H	STBIO MEDIA	110533
Rotary evaporator	EYELA	SB-1000
Sulfuric acid	Fluka	30743-2.5L-GL
TLC silica gel 60 F254	MERCK	1.05554.0001
Trifluoroacetic acid	Alfa Aesar	10229873
Ultrasonic vibration machine	DELTA	DC600