Raeesa Gupte, PhD
Capillary electrophoresis (CE) is a separation technique that was developed for the separation of charged molecules such as peptides and amino acids. The technique has now been optimized for a number of substances including DNA and drugs. Therefore, it is routinely used in forensics, disease diagnosis, and therapeutic drug monitoring.
How CE works
CE separates molecules based on charge, size, hydrophobicity, and stereospecificity. The CE apparatus consists of a buffer-filled fused silica capillary tube whose ends are connected to a high-voltage power supply. The sample is introduced at one end of the capillary and a detector placed at the other end reads the separated molecular peaks.
The small inner diameter of the capillary (10 – 100 mm) enables application of very high voltages and efficient heat dissipation. Capillary lengths of up to 20 cm allow separation at high resolution. Commercially available instruments are automated and use multiple capillaries for rapid, high-throughput separation of analytes using very small sample volumes.
Various types of CE have been developed by altering the capillary buffer composition as follows:
- Capillary zone electrophoresis (CZE): A low viscosity buffer fills the capillary and analytes in the sample are separated based on their charge to mass ratio. Compounds with a larger ratio migrate through the electric field faster and separate first.
- Capillary gel electrophoresis (CGE): The capillary contains a gel matrix such as bis-polyacrylamide, agarose, or methylcellulose. It is used to separate DNA, RNA, or proteins based on their molecular weight.
- Micellar electrokinetic chromatography (MEKC): A surfactant is added to the buffer solution to form micelles in order to separate neutral analytes. Hydrophobic compounds get trapped within the micelles and migrate slower, while hydrophilic compounds migrate through the solution fairly quickly.
- Capillary isoelectric focusing (CIEF): Ampholytes (substances that can act as an acid or a base in solution) added to the buffer solution create a pH gradient that is used to separate proteins and peptides according to their isoelectric point.
The same apparatus can be used for different types of CE, making it a versatile analytical technique.
Clinical applications of CE
Various biological samples including blood, urine, cerebrospinal fluid, and tissue lysates may be analyzed in clinical laboratories using CE. Of these, blood and urine have been extensively validated. Clinical applications of CE include:
Diagnosis of blood disorders
CZE can separate serum proteins into distinct bands of albumin, µ globulins, ß globulins, and γ globulin. The γ fraction provides information on disorders caused by clonal expansion of plasma cells. Monoclonal immunoglobulins produce a narrow band near the γ region. Large bands (> 3g/dL) of these monoclonal proteins are usually present in patients with multiple myeloma, whereas lower concentrations (< 3g/dL) occur in leukemia, lymphoma, amyloidosis, or monoclonal gammopathy of undetermined significance.
CZE can also be used to detect hemoglobinopathies. Hemoglobinopathies may be caused by mutations in the amino acid sequence resulting in structural hemoglobin (Hb) variants. Healthy adults express HbA and HbA2, whereas HbF is the primary hemoglobin produced by the fetus. Although over 800 hemoglobin variants have been identified, only a few of them are clinically significant. These include HbC, HbD, HbE, and the sickle cell disease-causing HbS. Another type of hemoglobinopathy, thalassemia, is caused by gene deletions or mutations that reduce production of the normal globin chain. CZE and CIEF have been used to qualitatively determine the presence of hemoglobin variants as well as to quantify globin chains in blood.
Hemoglobin analysis may also be used to diagnose diabetes. Chronically elevated circulating glucose levels result in glycation of HbA to form HbA1c. CIEF and commercially available CE instruments have been successfully used to determine the ratio of HbA1c to HbA as a measure of glycemic control in diabetic patients.
Therapeutic drug monitoring
Drugs with a narrow therapeutic index may be less effective or produce toxicity outside a certain concentration range. Therefore, the blood levels of such drugs need to be closely monitored. For instance, aminoglycoside antibiotics such as gentamycin, kanamycin, amikacin, and streptomycin are associated with the risk of ototoxicity and nephrotoxicity. CE with borate buffer at pH 10 forms UV-absorbing borate complexes that are used to measure antibiotic levels.
Blood levels also need to be monitored for drugs with considerable inter-individual pharmacokinetic variability and drug-enzyme interactions. For example, multiple drugs are often administered to treat epilepsy. Monitoring their levels is necessary to ensure patient compliance and prevent toxicity. Accordingly, CZE and MEKC have been used to measure phenytoin, carbamazepine, lamotrigine, and gabapentin.
Analgesics like aspirin, ibuprofen, and paracetamol sometimes need to be measured in the emergency room due to the risk of serious toxicity from overdose. Various CZE assays have been developed to detect these drugs and their metabolites in blood and urine.
CZE and MEKC is used in forensic toxicology to detect illicit substances or drugs of abuse in blood or urine. It has also been used in analysis of explosive compounds, gunshot residues, and inks and dyes. Sequencing DNA and short tandem repeats using CGE is legally permissible evidence of human identification.
When used in combination with polymerase chain reaction (PCR), CE has many applications as a molecular diagnostic tool. It is used to identify and characterize the microorganisms that cause infectious diseases in order to determine the best treatment strategies. It is used to identify gene polymorphisms associated with cancer diagnosis and prognosis. Several inherited genetic diseases including cystic fibrosis, fragile X, mitochondrial heteroplasmy, and spinocerebellar ataxia can also be detected by automated CGE and microfluidic gel electrophoresis.
Use of CGE, CZE, and CIEF for protein analysis in other biological matrices is also being investigated. Analysis of cerebrospinal fluid proteins can help in the diagnosis and management of neurological disorders and study of disease progression. Similarly, analyzing pleural effusions can help categorize them as transudates or exudates and guide therapeutic decisions.
The future of CE: Tiny but mighty
Due to its speed, versatility, and low cost, CE is often used as an alternative to more traditional analytical techniques such as liquid or gas chromatography. Miniaturized CE has the potential to further reduce speed and sample volumes while performing more complex and integrated analyses.
Microchip capillary electrophoresis, first developed in the early 1990s, is a miniaturized device with microfluidic chambers filled with various separation matrices. Microchip CE devices have been tested for diagnosis of cancer, cardiovascular, neurological, and infectious diseases. Several microchip CE devices are commercially available and being increasingly used for routine analysis. They promise an automated high-throughput approach with speedy analysis (typically seconds)—highly desirable features for any clinical laboratory. With further improvements in detection sensitivity of certain analytes, these lab-on-a-chip devices are likely to replace many slower and complex analytical systems currently in use.