출처: 박사의 실험실.B 질 벤턴 – 버지니아 대학
많은 화학 분석의 목표는 정량 분석이며, 시료의 물질의 양이 결정됩니다. 시료로부터 알 수 없는 농도를 정확하게 계산하기 위해서는 주의 깊은 시료 준비가 핵심이다. 샘플을 처리하거나 전송할 때마다 일부 샘플을 손실할 수 있습니다. 그러나 샘플 손실을 최소화하기 위한 전략이 있습니다. 또한 시료 손실에 대처하고 농도를 정확하게 측정하기 위한 전략도 있습니다.
샘플 손실을 최소화하기 위해 시료 처리 및 전송 단계 수를 최소화하는 것이 이상적입니다. 예를 들어, 솔리드 샘플을 플라스크에 직접 매스하여 용액이 전달 단계를 감소시키는 데 필요한 경우 한 플라스크에서 다른 플라스크로 전송해야 하고 희석이 이루어지고 있는 경우 유리 제품을 헹구면 모든 샘플이 전송되도록 합니다. 다른 전략은 샘플에 더 구체적입니다. 예를 들어, 단백질과 같은 유리에 흡착하는 샘플은 폴리프로필렌 일회용 튜브에서 더 잘 처리될 수 있습니다. 튜브는 친수성이 아니기 때문에 소량의 시료가 물에 배관될 경우 이미 튜브에 물을 첨가하여 샘플을 용매로 직접 배관할 수 있습니다. 수화 후 용성으로 인한 손실로 인해 샘플을 완전히 건조시키기보다는 농축하는 것이 더 좋을 수 있습니다.
샘플 손실의 또 다른 원인은 불완전한 샘플 조작을 통해서입니다. 예를 들어, 파생 프로시저가 사용되고 파생이 불완전한 경우 전체 양의 샘플이 관찰되지 않습니다. 이와 같은 오류는 체계적인 오류이며, 파생 절차 변경과 같은 문제를 수정하여 해결할 수 있다. 측정에서 체계적인 오류의 또 다른 원인은 매트릭스 효과입니다. 이들은 특정 물질의 측정을 방해하고 샘플이 효과를 줄일 수있는 샘플과 동일한 매트릭스에서 교정을 수행 할 수 있습니다.
정량 분석은 일반적으로 외부 또는 내부 표준을 사용하여 수행됩니다. 외부 표준의 경우, 교정 곡선은 관심 있는 별분의 상이한 공지된 농도를 측정하여 이루어집니다. 그런 다음 샘플은 표준과 별도로 실행됩니다. 내부 표준의 경우 표준은 관심 있는 단언과 동일한 샘플에 있으므로 동시에 측정을 수행할 수 있습니다. 전형적으로, 다른 종은 내부 표준이라고 불리며 그 내부 표준및 분석물의 반응 비율이 계산된다. 응답 계수라고 하는 응답 비율이 농도에 비례한다는 것이 아이디어입니다. 방법은 관심 분석기와 내부 표준을 구별할 수 있어야 하지만 내부 표준이 추가된 후에 발생하는 모든 샘플 손실은 두 물질 모두에 대해 유사해야 하므로 응답 비율은 동일하게 유지됩니다. 내부 표준을 사용하는 특별한 경우는 표준 추가 방법이며, 여기서 는 Aalyte의 양이 증가함에 따라 솔루션에 추가되고 원래의 양의 딜리바이트가 다시 계산됩니다. 내부 표준은 크로마토그래피, 전기화학 및 분광법에 사용될 수 있습니다.
Internal standards are used in many fields, including spectroscopy and chromatography. In spectroscopy, internal standards can help correct for random errors due to changes in light source intensity. If a lamp or other light source has variable power, it will affect the absorption and consequently, emission of a sample. However, the ratio of an internal standard to analyte will stay constant, even if the light source does not. One example of this is using lithium (Li) as an internal standard for the analysis of sodium in a blood sample by flame spectroscopy. Li is chemically similar to sodium but is not natively found in blood.
For chromatography, internal standards are often used in both gas chromatography and liquid chromatography. For applications with mass spectrometry as the detector, the internal standard can be an isotopically-labeled analyte, so that the molecular weight (MW) will be different than the analyte of interest. Internal standards are commonly used in pharmaceutical or environmental analyses.
Sample loss can occur every time a sample is handled or transferred, thereby making accurate calculations of concentration difficult.
To ensure accuracy, the effects of sample loss must be minimized using careful sample preparation and by limiting the number of sample handling and transfer steps. However, sample loss can also occur due to systematic errors, such as incomplete sample manipulation, matrix effects, and variations in analytic procedure.
These sources of loss can be accounted for by adding a known concentration of a species similar, but not identical, to the compound of interest. This is called an internal standard. Any sample losses that occur to the internal standard should be similar for the analyte, allowing for the concentration to be accurately calculated.
This video will illustrate the use of an internal standard and proper lab technique to account for sample loss when determining the concentration of an unknown.
An internal standard is a substance added in a known amount to standards, samples, and blanks during an analysis.
In chromatography and spectroscopy, the ratio of the signal for the internal standard and the analyte is calculated. This ratio, called the response factor, is proportional to the ratio of the analyte and standard concentrations.
Response factor, R, can be expressed by the following equation, where A represents the analytical signals of the sample and internal standard and C represents the concentrations of the sample and internal standard.
An internal standard can compensate for both systematic and random errors. For example, random errors—such as inconsistencies when measuring a sample—will be the same for both the internal standard and the analyte. Therefore, the ratio of their signals will not change.
For systematic errors, such as matrix effects in solution, the ratio will be unaffected as long as the matrix effect is equal for both the standard and the analyte.
While internal standards provide great benefit, it can be difficult to choose one that is suitable. An internal standard must have a signal that is similar, but not identical, to the analyte. It also cannot affect the measurement of the analyte in any way.
Finally, the concentration must be well known. This is achieved by ensuring that the internal standard is not natively present in the sample; thus, the only source of it in solution is the known concentration added.
In the following experiment, the concentration of caffeine in an unknown sample will be determined by gas chromatography.
This is achieved by creating a calibration curve using known caffeine solutions, with adenine as the internal standard. The slope of the calibration curve is equal to the response factor.
Once the response factor is known, the concentration of the unknown can be calculated from its measured chromatogram area ratio.
Now that you understand the basics of internal standards, let’s take a look at the procedure.
To begin the procedure, accurately weigh 100 mg of the internal standard, adenine, into a clean beaker.
Next, dissolve it in roughly 20 mL of dimethyl sulfoxide, and mix the solution.
Once the adenine has dissolved, pour the solution into a 50-mL volumetric flask.
Rinse the beaker and stir bar with 10 mL of DMSO, and pour the rinse into the flask. Repeat this rinse twice, to ensure proper solution transfer. Fill to the calibration mark, resulting in an internal standard with a concentration of 2 mg/mL.
Next, weigh 100 mg of caffeine into a beaker to prepare a stock solution. Dissolve the caffeine with a small amount of methanol. Then, use 3 rinses to transfer this solution to a fresh 25 mL volumetric flask. This is the 4 mg/mL stock solution. Use it to create 3 caffeine standards.
Next, add 0.2 mL of the internal standard, adenine, to each flask. Fill each to the final volume with methanol. Transfer each solution to a sample vial.
Run each caffeine standard through a gas chromatograph. Calculate the ratio of peak areas for the caffeine versus the adenine standard.
First, weigh 2 g of coffee into a 100-mL beaker, and record the weight.
Next, add 20 mL of methanol to extract the caffeine from the coffee. Allow the solution to stir for 20 min.
Using a Büchner funnel, filter out the coffee grounds. Rinse the beaker with a small amount of methanol, and pour this rinse into the funnel. Repeat the rinse twice.
Measure the final volume of the filtrate; it should be approximately 35 mL.
To prepare the sample for analysis, add 1 mL of the coffee extract to a sample vial. Then, add 0.2 mL of the adenine internal standard, and place the vial into the instrument’s auto-sampler rack.
Run a gas chromatography analysis of the sample, ensuring that the conditions are such that the caffeine and adenine are separate.
After completing the analysis, compute the peak area for both the internal standard and the analyte.
Once all the samples have been analyzed, the standard calibration curve can be determined for the caffeine/adenine solutions by plotting the ratios of the peak areas versus the ratios of the concentrations. The slope of this line, which represents the response factor, was 1.8.
Next, the GC data from the extracted coffee sample is analyzed. The ratio of the peak areas was calculated to be 1.78. Using the response factor and the known concentration of the internal standard, adenine, the concentration of caffeine in the unknown sample was calculated to be 0.33 mg/mL.
Many different types of reactions, across various scientific disciples, utilize internal standards to minimize the effects of errors and sample loss.
The effects of sample loss encountered during sample preparation can be minimized using internal standards, keeping their concentration ratio nearly constant.
In this example, bioactive lipids were extracted from lysed cells using a liquid-liquid extraction process. Stable isotope internal standards were added at the beginning of extraction to account for errors during sample preparation.
Internal standards were not only critical for the preparation of the bioactive lipids, but also for the analysis. The lipids were separated using high-performance liquid chromatography, and analyzed via mass spectrometry.
In spectroscopy, internal standards can help correct for random errors due to changes in light source intensity. If a lamp or other light source has variable power, it will affect the absorption and consequently, emission of a sample. However, the ratio of an internal standard to analyte will stay constant, even if the light source does not.
In chromatography, one of the largest sources of error is the injection. Auto-samplers help minimize this, but error can still be 1–2% relative standard deviation.
In this example, vapor standards containing an internal standard were analyzed using gas chromatography to establish a calibration curve. Once this was complete, the unknown sample could then be measured and the losses due to volatility of the sample accounted for.
You’ve just watched JoVE’s introduction to internal standards. You should now understand best practices for minimizing sample loss, internal standards, and response factors.
Thanks for watching!
